EP0886679B1 - Parapoxviruses containing foreign dna, their production and their use in vaccines - Google Patents
Parapoxviruses containing foreign dna, their production and their use in vaccines Download PDFInfo
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- EP0886679B1 EP0886679B1 EP97904453A EP97904453A EP0886679B1 EP 0886679 B1 EP0886679 B1 EP 0886679B1 EP 97904453 A EP97904453 A EP 97904453A EP 97904453 A EP97904453 A EP 97904453A EP 0886679 B1 EP0886679 B1 EP 0886679B1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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- C12N2710/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2710/24243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to recombinant parapoxviruses, their preparation, vaccines and immunomodulators containing them.
- the genetically modified parapoxviruses according to the invention carry deletions and / or insertions in their genome.
- the deletion of genomic sections of the Parapockenviren and / or the insertion of foreign DNA can lead to the reduction or loss of their pathogenicity (attenuation).
- Insertion inserts genetic information from pathogens or biologically active substances into the genome of the parapox viruses. These foreign genetic information is expressed as part of the recombinant parapochenviruses, for example in cell cultures, tissues or in intact organisms.
- parapox viruses are e.g. used in vaccines or immunomodulators.
- Expression of the foreign DNA in the genome of the parapoxviruses triggers e.g. in the vaccine a defense reaction against the pathogens, which are represented by the foreign genetic information.
- the non-specific immunity of the vaccine can be stimulated.
- parapox viruses is abbreviated by PPV.
- PPV itself can have an immunomodulatory effect as it stimulates non-pathogen-specific immune reactions in the organism.
- Parapox virus preparations for example, are used successfully in veterinary medicine to increase general defenses
- Vaccines which are pathogen-specific require several days to weeks depending on the antigen for the establishment of the protection, but then provide a long, lasting from months to years protection.
- Vaccines produced on the basis of recombinant parapoxviruses can thus be used as biological products for improved control of infectious diseases, since they both build a long-lasting, pathogen-specific immunity and induce a very rapidly occurring pathogen-specific protection in the organism.
- parapoxvirus representatives isolated from camels, red deer, chamois, seals, seals and sea lions are also described. Whether it is a stand-alone species within the genus Parapox viruses or isolates of the species described above, is not yet finally resolved.
- Ref. # 1 gives an overview of the clinical pictures described so far. The fight against the diseases is possible through the use of prophylactic measures, such as vaccines. However, the vaccines available so far, which were developed exclusively on the basis of Parapopxvirus ovis, show unsatisfactory activity (Ref. # 2).
- the object of the invention is to utilize the PPV as a carrier (vector) of foreign genetic information which is expressed.
- PPV Vectors based on avipox, raccoonpox, capripox, swinepox or vaccinia virus have already been described as vectors for the expression of foreign genetic information. The insights gained can not be transferred to PPV. As comparative studies have shown, there are morphological, structural and genetic differences between the individual genera of the poxviruses. For example, the PPV can be differentiated from the other genera of the pox viruses with serological methods, which is due to different protein patterns and thus to different genetic information. For example, some representatives of the poxviruses have the ability to agglutinate erythrocytes. This activity is mediated by a surface protein, the so-called haemagglutinin (HA). PPVs do not have this activity.
- HA haemagglutinin
- VEGF vascular endothelial growth factor
- various mammalian species eg mouse, rat, guinea pig, bovine, human.
- Seq. ID No: 1 shows the nucleotide sequence of the gene in D1701
- Seq. ID No: 15 is the amino acid sequence of the corresponding protein of D1701.
- a homologous gene has also been described in the PPV strains NZ2 and NZ7 (ref. # 6), but its function is not known.
- a corresponding gene is present in other poxviruses, e.g. Orthopoxviruses, not known. Later in the text, this gene is called the VEGF gene.
- ORF3 because of its similarity to a gene in PPV NZ2 (identity 76%, similarity 83%).
- Seq. ID No: 4 shows the nucleotide sequence of the gene at D1701. In the further course of the text, this gene is called ORF3 gene.
- all PPV species are suitable for carrying out the present invention.
- virus strains which can be multiplied to titer> 10 5 PFU (plaque forming unit) / ml in a tissue culture and can be purified from the medium of the infected cells as an extracellular, infectious virus.
- Preference is given to the genus of PPV the species PPV ovis (Orf viruses).
- the multiplication of the viruses takes place in a customary manner in tissue cultures of animal cells, such as mammalian cells, for example in ovine cells, bovine cells, preferably in bovine cells such as the permanent bovine kidney cell BK-K1-3A (or its progeny) or monkey cells such as the permanent monkey kidney cells MA104 or Vero (or their progeny).
- animal cells such as mammalian cells, for example in ovine cells, bovine cells, preferably in bovine cells such as the permanent bovine kidney cell BK-K1-3A (or its progeny) or monkey cells such as the permanent monkey kidney cells MA104 or Vero (or their progeny).
- Propagation is carried out in a manner known per se in stationary, roller or carrier cultures in the form of closed cell aggregates or in suspension cultures.
- the cells and cell turfs used for the multiplication of the viruses are increased in the usual way almost to confluence or up to optimal cell density.
- the multiplication of the viruses takes place with or without the addition of animal serums.
- serum this is added to the propagation medium in a concentration of 1-30 vol .-%, preferably 1-10 vol .-%.
- Infection and virus multiplication takes place at temperatures between room temperature and 40 ° C, preferably between 32 and 39 ° C, more preferably at 37 ° C for several days, preferably until complete destruction of the infected cells.
- cell-bound virus can be mechanically or by ultrasound or mild enzymatic proteolysis e.g. be additionally released with trypsin.
- the virus-containing medium of the infected cells can then be further worked up, e.g. by removing the cell debris by filtration with pore sizes of e.g. 0.2-0.45 ⁇ m and / or low speed centrifugation.
- Filtrate or centrifugation supernatant can be used for virus replenishment and purification.
- the filtrate or supernatant of a high-speed centrifugation are subjected to sedimentation of the virus particles.
- further purification steps can be connected by, for example, centrifugation in a density gradient.
- the non-essential gene may be partially or completely deleted from the genome of the PPV.
- restriction enzyme recognition sites can serve as insertion sites.
- the protein kinase gene (PK gene) of PPV D 1701 called.
- the PK gene is expressed late in the reproductive cycle of the virus.
- the identification of the PK gene on the genome of PPV can be done using the versions of the DNA sequence shown in Sequence Listing ID No: 2, No: 9 or No: 13.
- the region of the gene can be found, for example, by hybridization experiments, genome sequence analyzes and / or polymerase chain reactions on the genome of a PPV.
- the essential gene can be partially or completely removed from the genome of the PPV.
- Genome segments which do not code for functional gene products and have no essential regulatory functions are in principle suitable as insertion sites for foreign DNA. Particularly suitable are areas with repetitive sequences, since changes in partial areas can be compensated by remaining Sequenzrepetitionen. This includes genes that occur in two or more copies, so-called gene duplications.
- insertion sites on the genome of PPV are also found via modifications of the viral genome sequences. Genomic sites where nucleotide exchanges, deletions and / or insertions or combinations thereof do not block viral replication represent potential sites of insertion. To test whether a potential site of insertion is a suitable insertion site, foreign DNA is introduced into the potential by known molecular biology techniques Insertion site inserted and examined the resulting virus mutant for viability. If the virus recombinant is capable of replication, the site of interest is an appropriate insertion site.
- the genome of the PPV is first purified. Starting from the virus prepared according to 1 (above), the genome is isolated and purified. The extraction of native viral DNA is preferably carried out by treating the purified virions with aqueous solutions of detergents and proteases.
- Detergents include anionic, cationic, amphoteric, nonionic detergents.
- ionic detergents are used.
- Particularly preferred are sodium dodecyl sulfate, sodium lauryl sulfate.
- proteases may be mentioned all proteases which operate in the presence of detergent, e.g. Proteinase K and pronase. Preference is given to proteinase K.
- Detergents are used in concentrations of 0.1-10 vol .-%, preferably 0.5-3 vol .-%.
- Proteases are used in concentrations of 0.01-10 mg / ml virus lysate, preferably 0.05-0.5 mg / ml virus lysate.
- buffer substances may be mentioned: salts of weak acids with strong bases such as e.g. Tris (hydroxymethylaminomethane), salts of strong acids with weak bases, e.g. primary phosphates or mixtures thereof.
- the following buffer system may preferably be mentioned: tris (hydroxymethylaminomethane).
- the buffer substances or buffer systems are used in concentrations which ensure pH values at which the DNA does not denature. Preference is given to pH values of 5-9, particularly preferably 6-8.5, completely particularly preferred 7-8, in particular work in the neutral range is called.
- DNase inhibitors are e.g. Ethylenediaminetetraacetic acid in concentrations of 0.1-10 mM (millimoles), preferably about 1 mM.
- the extractants used are solvents such as phenol, chloroform, isoamyl alcohol or mixtures thereof.
- solvents such as phenol, chloroform, isoamyl alcohol or mixtures thereof.
- a mixture of phenol and chloroform / isoamyl alcohol is first used, wherein the extraction takes place in one or more stages.
- the DNA so extracted is preferably extracted from the aqueous solution with e.g. Alcohol, preferably with ethanol or isopropanol and with the addition of monovalent salts such.
- Alkali chlorides or acetates preferably lithium chloride, sodium chloride or sodium acetate, potassium acetate, precipitated (see loc cit.).
- the purified viral DNA is now used to produce DNA fragments. It is treated for this purpose, for example, with restriction enzymes. Suitable restriction enzymes are, for example, Eco RI, BamHI Hin dIII, KpnI.
- genome fragments can be synthesized by polymerase chain reaction (PCR).
- primers are selected from already known sequence segments of the virus genome and synthesized by means of, for example, Taq or Pfu polymerase of the primer pair-limited genome section in vitro.
- DNA fragments resulting from restriction digestion or PCR can be cloned into vector systems according to the methods described in (Sambrock 89) become.
- plasmid vectors for example, depending on the size of the particular DNA fragment to be cloned, plasmid vectors, lambda phage vectors or cosmid vectors are suitable.
- Genomic fragments cloned into vectors are first analyzed by sequencing.
- the inserted DNA fragments are mapped by means of various restriction enzymes and suitable subfragments are cloned into plasmid vectors.
- the sequencing reaction is carried out, for example, using the T7 sequencing kit from Pharmacia according to the manufacturer.
- the double-stranded plasmid DNA required for this purpose is preferably prepared by the PEG method (Hattori and Sakaki 1985).
- CGC computer analysis
- ORF open reading frames
- the identified ORFs are characterized by detection of their respective corresponding transcripts in virus-infected cells.
- the total RNA of virus-infected cells is isolated, for example, by the AGPC method (Chomczynski and Sacchi, (20) 1987).
- the specific transcripts can be identified together with their 5 'and 3' ends.
- the gene can be destroyed by gene disruption or gene deletion. In this case, either the entire gene or parts thereof are removed from the PPV genome or the reading frame of the gene is interrupted by the insertion of foreign gene sequences. The resulting virus is tested for reproductive ability. Can the virus even without the Replicating the existence of the destroyed gene, this is a non-essential gene.
- the corresponding Regions of the PPV genome which include the insertion sites isolated.
- the corresponding regions of the PPV genome are cloned.
- the genomic fragments prepared above are inserted into bacterial or eukaryotic vectors.
- Particularly preferred are first bacterial plasmid or phage vectors.
- double-stranded plasmid or phage vector DNA molecules are treated with restriction enzymes to form suitable ends for insertion.
- Plasmids are known plasmids, e.g. pBR322 and its derivatives, e.g. pSPT18 / 19, pAT153, pACYC184, pUC18 / 19, pSP64 / 65.
- phage vectors are used e.g. the known variants of the phage lambda, e.g. -ZAP, -gt 10/11 or phage M13mp18 / 19.
- the restriction enzyme treated plasmid or phage vector is incubated with an excess of the DNA fragment to be inserted, e.g. in the ratio 5: 1, mixed and treated by means of DNA ligases, ligated into the vector.
- the ligation mixture is introduced into and propagates the plasmids or phages in prokaryotic or eukaryotic cells, preferably in bacteria (e.g., Escherichia coli strain K-12 and its derivatives).
- Transformation and selection of the bacteria is carried out as described in Molecular Cloning loc. sit. described.
- the identity of the foreign DNA is preferably verified via hybridization experiments and more preferably via sequence analyzes. If necessary, subcloning is carried out.
- the gene encoding the protein kinase is localized on the genome of the PPV and subsequently isolated in part or all with its flanking genome segments.
- This area can be described as for the cloning of the VEGF gene. on the fragmentation of the PPV genome (cleavage sites see Fig. 1). preferably with subsequent cloning of the fragments, and selection of the fragments or clones which contain parts of the PK gene or preferably the entire PK gene with flanking DNA sequences of the PPV genome are obtained.
- DNA molecules used as primers of a PCR or as Probes can be used for a hybridization, can be derived from the sequence of Protocol ID No: 2 or ID No: 9.
- the gene encoding the 10kDa protein is located on the genome of the PPV and partially or preferably entirely with its flanking PPV, according to the procedure detailed for the VEGF gene and the PK gene (above) Genome sequences isolated.
- the region of the PPV genome containing the gene for the 10kDa protein or parts thereof is preferably obtained by PCR and / or cloning and identification and selection of the appropriate clones.
- DNA molecules that can be used as primers of a PCR or as probes for hybridization can be found in reference # 8. If necessary, subcloning is carried out.
- the procedure corresponds to the detailed cloning of the VEGF gene (above).
- the DNA molecules that can be used to isolate the corresponding regions as primers of a PCR or as probes for hybridization are described in the Sequence Protocol ID No: 4 (ITR region) and No: 7 (region between PK gene and HDIR Gene).
- insertion plasmids Based on the localized and cloned genomic fragments of PPV identified in accordance with Section 2, so-called insertion plasmids are produced, which can be used to insert foreign DNA into the genome of the PPV.
- the insertion plasmids carry the foreign DNA to be inserted into the PPV flanked by genome segments of the PPV.
- Synthesized oligonucleotides carrying new unique restriction enzyme cleavage sites can be incorporated into these.
- the resulting plasmids are propagated and selected as described above.
- the identified and / or produced unique restriction enzyme recognition sites serve to insert foreign DNA into the PPV genome.
- the insertion of the foreign DNA is carried out by known methods (Ref. # 11).
- the deletion of subfragments from the cloned PPV genome fragments can be carried out, for example, by treatment with restriction enzymes which preferably have more than one, particularly preferably 2, recognition sites have. After the enzyme treatment, the fragments obtained are isolated, as described above, for example by electrophoresis, and the corresponding fragments are reassembled by ligase treatment. The resulting plasmids are propagated and the deleted plasmids are selected.
- a unique restriction enzyme recognition site on the PPV genomic fragment is used as a starting point for bidirectional degradation of the fragments with an endonuclease, for example the enzyme Bal31.
- the size of the deletion can be determined by the duration of the action of the enzyme and checked by gel electrophoresis.
- Synthetic oligonucleotides, as described under 3.1 (above), are ligated to the newly formed fragment ends.
- the gpt selection system is used, which is based on the guanyl phosphoribosyl transferase gene of E. coli. This gene, expressed in a eukaryotic cell, mediates resistance to mycophenolic acid, an inhibitor of purine metabolism. Its use in the construction of recombinant vector viruses has been described several times (see reference # 16 / # 17).
- Confluent adult BK-KL-3A cells are infected with an infection dose of 0.001 to 5, preferably 0.1 MOI (multiplicity of infection). Two hours later, the infected cells are, for example, with DNA (2-10 ug) of the plasmid pMT-10 either by the CaPO 4 glycerol shock method or using a transfection kit according to the manufacturer (DOSPER, Boehringer-Manaheim) transfected. Subsequently, these cell cultures are incubated with medium for 3 to 6 days at 37 ° C, 5% CO 2 atmosphere until cpe or plaque formation is visible.
- the DNA is obtained from the virus in question and hybridized with nucleic acid which is identical at least in part to the inserted foreign DNA.
- the individual plaque-purified and identified as recombinants PPV are preferably tested again for the presence and / or expression of the foreign DNA.
- Recombinant PPV stably containing and / or expressing the foreign DNA are available for further use.
- the expression of the foreign DNA at the protein level can be detected by, for example, infection of cells with the virus and subsequent immunofluorescence analysis with specific antibodies to that of the Foreign DNA encodes protein or by immunoprecipitation or Western blotting with antibodies against the foreign DNA encoded protein from the lysates of infected cells.
- RNA is isolated from virus-infected cells and hybridized with a DNA probe which is at least partially identical to the inserted foreign DNA.
- Suitable genomic fragments are contained on HindIII fragment I of PPV ovis strain D1701 (and its respective derivatives) (Sequence Protocol ID No: 8 and ID No: 12).
- the resulting DNA fragments were separated by agarose gel electrophoresis after cleavage of purified viral DNA with the Hind III restriction enzyme, the excised fragment of about 5.6 kbp and I by means of the Qiaex ® method (Qiagen) and purified.
- This DNA fragment was cloned by standard techniques (Maniatis et al.) Into HindIII digested and CIP (calf intestinal phosphatase) treated vector plasmid pSPT18 (Boehringer, Mannheim).
- the resulting recombinant plasmids pORF-1 and pORF-2 differ only in the orientation of the insert.
- the preparation of a restriction map allowed further subcloning, as shown in FIG. All recombinant plasmid DNAs were tested by Southern blot hybridization for restriction digested viral or plasmid DNA to verify their identity and viral origin.
- DNA sequencing was accomplished by using double stranded DNA of the various recombinant plasmids and SP6 and T7 specific primers that bind to both ends of the cloning site of the vector plasmid pSPT18.
- the Sanger dideoxy chain termination method was performed according to the manufacturer's recommendations (Pharmacia Biotech) in the presence of 35 S- [ ⁇ ] -dATP and T7 DNA polymerase.
- a plurality of oligonucleotides were synthesized and used for sequencing both strands of HindIII fragment I.
- 7-deaza-GTP was used and the sequencing products separated, if necessary, in formamide containing denaturing polyacrylamide gels.
- the plasmid pGSRZ was constructed which contains the functional LacZ gene of E. coli under the control of the vaccinia virus 11K promoter.
- the relevant DNA parts of the plasmid pUCIILZ (see reference # 7) were isolated and cloned into the plasmid pSPT18.
- This functional 11K-LacZ gene combination (hereinafter referred to as LacZ cassette) can be obtained by isolating a 3.2 kbp SmaI-SalI fragment from pGSRZ ( Figure 3).
- Plasmids pMT-2 and pMT-4 were prepared by removing the LacZ gene from p18Z (obtained by insertion of the BamHI fragment from pGSRZ into pSPT18) or the gpt gene from pMT-5 by BamHI digestion, and into pMT-1 was inserted (Figure 7).
- the functional gpt gene was amplified by PCR from the gpt plasmid pMSG (Pharmacia Biotech) and cloned into the vector pCRII by so-called TA cloning according to the instructions of the manufacturer (Invitrogen Inc.).
- double selection cassettes could be constructed which in the combinations and orientations shown express the LacZ gene or gpt gene under the control of the 11K promoter and / or the P VEGF .
- Example XX LacZ-VEGF deletion
- YY intergenic Ba131
- these selection cassettes can be inserted into the various PPV orf DNA plasmids after appropriate restriction enzyme cleavage and isolation.
- the plasmid pMT-10 was constructed.
- the functional LacZ and gpt genes were substituted for the deleted VEGF ORF.
- the activity of the LacZ gene was demonstrated in PPV D1701-infected cells (not shown) so that further selection was made for gpt- and lacZ-expressing VEGF deletion mutants of the D1701.
- the plasmid pORF-PB ( Figure 1) contains a single NruI site located between the protein kinase (F10L) and the HD1R gene. After linearization of pORF-PB with this restriction enzyme, the LacZ cassette (after fill-in reaction) was blunt-end ligated. Recombinant plasmids containing the functional LacZ gene were cleaved, e.g. was selected with BglI and the correct insertion demonstrated by Southern blot hybridization with a LacZ-specific probe as well as by partial DNA sequencing of the LacZ and PPV DNA transition.
- PCR mutagenesis To introduce a novel, unique restriction site into any desired location of cloned PPV DNA fragments, the technique of PCR mutagenesis can be used (see Fig. 10, ref. # 12).
- two PCR reactions are carried out separately using the primer pair E1 + EV2 (PCR A) and EV1 + XB (PCR B), respectively. All primers cover 25 nucleotides that are identical to the PPV DNA sequence at the indicated sequence positions.
- primer XB contains the authentic sequence, e.g. representing the XbaI site ( Figure 1), a new EcoRI site was inserted into primer E1 at the 5 'end and a new EcoRV site was inserted into primers EV1 and EV2 (which are complementary to each other).
- the EcoRV site which is absent in the entire sequence of pORF-1 or -2, was placed at the site chosen for the introduction of the LacZ cassette.
- the PCR products obtained from reactions A and B are purified, denatured to single strands and mixed together under reassociation conditions; then they are used for the last PCR C reaction.
- primers E1 and XB are now used to extend in this example the left 793 bp of pORF-1.
- the resulting PCR product from reaction C is cleaved with EcoRI and XbaI and then ligated to the EcoRI and XbaI digested plasmid pORF-XB.
- the plasmid pORF-1EV ( Figure 5) contains the EcoRV restriction site at the desired position; it can then be used for linearization and ligation with the LacZ cassette.
- mismatch primers containing defined base changes or a single base deletion may be used for the method described in this example to generate, for example, translation stop codons or amino acid deletions at any desired location in the viral DNA sequence.
- New or additional sequences can be introduced into the coding sequences of one of the described ORFs after cleavage to restriction sites which occur only once in the selected gene.
- the unique XcmI site located in the right-hand part of the gene homolog F10L, was used to linearize the plasmid pORF-1. Both the LacZ cassette and the cleaved pORF-1 DNA were blunt-ended by T4 or Klenow DNA polymerase, and competent E. coli bacteria (DHS ⁇ F ') were used for transformation. The resulting bacterial colonies were tested for positive recombinant plasmids by colony filter hybridization using a LacZ-specific probe, and then the respective plasmid DNAs were subjected to restriction digestion.
- the VEGF coding region contains a single StyI site used as described above to insert the LacZ cassette.
- HindIII DNA fragment I By using restriction enzymes, defined portions of HindIII DNA fragment I are deleted to replace the deleted viral sequences by inserting foreign genes or portions thereof. This was done by cleavage of the plasmid pORF-PA with the restriction enzyme NruI (at a position in the ITR region, Fig. 1), wherein a 396 bp fragment was deleted. After a padding reaction, the LacZ cassette was ligated as described above. Illustration Figure 4 shows schematically the deletion of the 396 bp fragment from pORF-PA and the insertion of the LacZ cassette. Figures 5 and 6 show the thus constructed deletion / insertion plasmids pCE4 and pCE9 derived from pORF-PA.
- RNA analyzes such as Northern blot hybridization, to test the expression of potential VEGF genes in other PPV strains.
- viruses obtained in (a) or (b) are used as described above for the infection of BK-KL-3A and subjected to at least two further plaque titrations and purifications until a 100% homogeneous recombinant virus population is present.
- the D1701 VEGF gene is an early gene, the specific mRNA is transcribed in large quantities in D1701 virus-infected cells from two to four hours after infection at later times of infection. Therefore, the identified promoter region of the D1701 VEGF should be very useful for directing the expression of foreign genes or portions thereof in recombinant PPV viruses.
- the sequence comprising the VEGF promoter (35-40 nucleotides; Sequence Protocol ID No: 6) can be isolated by (i) PCR using the appropriate primers flanking the promoter region, (ii) subcloning the corresponding DNA sequence. Fragments or (iii) synthesizing the promoter sequence as an oligonucleotide. After linking the VEGF promoter to any gene of interest or to a DNA sequence, the resulting gene cassette can be used to produce recombinant PPV by any method as described in the preceding chapter.
- DNA sequencing of both DNA strands of the insert of pJS-1 demonstrated the presence of the 10kDa-specific sequence. Thereafter, D1701 encodes a 91 amino acid 10kDa protein that belongs to the of the PPV strain NZ-2 93.3% identical or 96.7% similar amino acids.
- the plasmids pDE-E1 and pRZ-E1 which contain the 4.25 kbp EcoRI fragment E of D1701 are used for the production of insertion and deletion plasmids in the 10 kDa gene (in principle as described above).
- the HindIII site (fragments KG) in the N-terminal portion of the 10kDa gene of D1701 (# 124- # 129 Sequence ID No: 11) can be used both for direct insertion of foreign DNA and for deletion (see Ba131 bidirectional digestion). of the 10 kDa gene.
- the second HindIII site (the multiple cloning site of the vector plasmid pSPT18) was removed in plasmid pDE-E1.
- pSPT18 DNA was digested with HindIII, the HindIII site disrupted by Klenow treatment and religated.
- the 4.25 kbp EcoRI fragment E of D1701 was then cloned into the EcoRI site of this new vector plasmid pSPT18dH.
- the resulting plasmid pRZ-E1 ( Figure 8) now has a unique HindIII site in the 10 kDa gene, which allows further simple manipulation.
- Sequence ID No: 1 of the application shows the VEGF gene located on HindIII fragment I of strain PPV D1701.
- Earyl promoter Nucleotides 50 to 64 mRNA start: Nucleotides 78 and 80, respectively mRNA Stop: Nucleotides 498 to 500 Translation start: Nucleotides 92 to 94 Translation stop: Nucleotides 488 to 490
- Sequence ID No: 2 of the application shows the protein kinase gene F10L (version 1) located on HindIII fragment I of strain PPV D1701
- Late promoter Nucleotides 48 to 66 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1738 to 1740
- Sequence ID No: 3 of the application shows the HD1R segment located on Hindin fragment I of strain PPV D1701.
- Sequence ID No: 4 of the application shows the ITR region located on HindIII fragment I of strain PPV D1701 and the ORF3 gene in this region.
- Sequence ID No: 5 of the application shows the F9L gene homolog located on HindIII fragment I of strain PPV D1701 (version 1).
- Sequence ID No: 6 of the application shows the VEGF promoter region located on HindIII fragment I of strain PPV D1701.
- Sequence ID No: 7 of the application shows the intergenic region located on HindIII fragment I of strain PPV D1701, located between the HD1R and the PKF10L gene.
- Putative translation stop HD1R Nucleotides 25 to 27
- Translation start PKF10L Nucleotides 223 to 225
- Sequence ID No: 8 of the application shows the complete nucleotide sequence of HindIII fragment I (version 1) of PPV strain D1701.
- Sequence ID No: 9 of the application shows version 2 of the protein kinase F10L gene located on HindIII fragment I of strain PPV D1701.
- Late promoter Nucleotides 48 to 66 RNA start signal: Nucleotides 72 to 80 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1585-1588
- Sequence ID No: 10 of the application shows version 2 of the F9L gene homolog located on HindIII fragment I of strain PPV D1701.
- Translation start Nucleotides 50 to 52
- Translation stop Nucleotides 722 to 724
- Sequence ID No: 11 of the application shows the 10 kDa gene located on EcoRI fragment E of strain PPV D1701.
- Translation start Nucleotides 5 to 7
- Translation stop Nucleotides 275 to 277
- Sequence ID No: 12 of the application shows the complete nucleotide sequence of version 2 of HindIII fragment I of PPV strain D1741.
- Sequence ID No: 13 of the application shows version 3 of the protein kinase FIOL gene located on HindIII fragment I of strain PPV D1701.
- Late promoter Nucleotides 48 to 66 RNA start signal: Nucleotides 72 to 80 mRNA start: Nucleotides 74 to 78 Translation start: Nucleotides 94 to 96 Translation stop: Nucleotides 1585-1588
- Sequence ID No: 14 shows the amino acid sequence of the PPV D1701 protein kinase F10L homolog (derived from Sequence ID No: 13).
- Sequence ID No: 15 shows the amino acid sequence of the PPV D1701 VEGF homolog (derived from Sequence ID No: 1).
- Sequence ID No: 16 shows the amino acid sequence of the PPV D1701 F9L homolog (derived from Sequence ID No: 10).
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Abstract
Description
Gegenstand der vorliegenden Erfindung sind rekombinante Parapockenviren, ihre Herstellung, Impfstoffe und Immunmodulatoren, die sie enthalten.The present invention relates to recombinant parapoxviruses, their preparation, vaccines and immunomodulators containing them.
Die erfindungsgemäßen gentechnisch veränderten Parapockenviren tragen in ihrem Genom Deletionen und/oder Insertionen. Die Deletion von Genomabschnitten der Parapockenviren und/oder die Insertion von Fremd-DNA kann zur Reduktion bzw. zum Verlust ihrer Pathogenität (Attenuierung) führen. Durch Insertionen werden Erbinformationen von Erregern oder biologisch wirksamen Substanzen in das Genom der Parapockenviren eingebaut. Diese fremden Erbinformationen werden als Bestandteil der rekombinanten Parapochenviren, beispielsweise in Zellkulturen, Geweben oder in intakten Organismen, zur Expression gebracht.The genetically modified parapoxviruses according to the invention carry deletions and / or insertions in their genome. The deletion of genomic sections of the Parapockenviren and / or the insertion of foreign DNA can lead to the reduction or loss of their pathogenicity (attenuation). Insertion inserts genetic information from pathogens or biologically active substances into the genome of the parapox viruses. These foreign genetic information is expressed as part of the recombinant parapochenviruses, for example in cell cultures, tissues or in intact organisms.
Die erfindungsgemäß hergestellten rekombinanten Parapockenviren werden z.B. in Vakzinen oder Immunmodulatoren eingesetzt. Die Expression der Fremd-DNA im Genom der Parapockenviren löst z.B. im Impfling eine Abwehrreaktion gegen die Erreger aus, die durch die fremde Erbinformation repräsentiert werden. Zusätzlich können die nicht spezifischen Abwehrkräfte des Impflings stimuliert werden. (Im folgenden wird der Begriff Parapockenviren durch PPV abgekürzt.)The recombinant parapoxviruses produced according to the invention are e.g. used in vaccines or immunomodulators. Expression of the foreign DNA in the genome of the parapoxviruses triggers e.g. in the vaccine a defense reaction against the pathogens, which are represented by the foreign genetic information. In addition, the non-specific immunity of the vaccine can be stimulated. (In the following, the term parapox viruses is abbreviated by PPV.)
PPV selbst können immunmodulatorisch wirken, da sie nicht-erregerspezifische Immunreaktionen im Organismus stimulieren. So werden Präparationen aus Parapockenviren beispielsweise in der Tiermedizin zur Steigerung der allgemeinen Abwehrkräfte erfolgreich eingesetztPPV itself can have an immunomodulatory effect as it stimulates non-pathogen-specific immune reactions in the organism. Parapox virus preparations, for example, are used successfully in veterinary medicine to increase general defenses
Vakzinen, die erregerspezifisch wirken, benötigen in Abhängigkeit des Antigens für die Etablierung des Schutzes mehrere Tage bis Wochen, bieten aber dann einen langen, über Monate bis Jahre anhaltenden Schutz.Vaccines which are pathogen-specific require several days to weeks depending on the antigen for the establishment of the protection, but then provide a long, lasting from months to years protection.
Vakzinen, die auf Basis von rekombinanten Parapockenviren hergestellt werden, können somit als biologische Produkte für eine verbesserte Bekämpfung von Infektionskrankheiten eingesetzt werden, da sie sowohl eine langanhaltende, erregerspezifische Immunität aufbauen als auch einen sehr schnell eintretenden erregerunspezifischen Schutz im Organismus induzieren.Vaccines produced on the basis of recombinant parapoxviruses can thus be used as biological products for improved control of infectious diseases, since they both build a long-lasting, pathogen-specific immunity and induce a very rapidly occurring pathogen-specific protection in the organism.
Die Kombination der immunstimulierenden Eigenschaften der PPV mit der Expression von Fremdantigenen, die einen homologen und/oder heterologen erregerspezifischen Schutz induzieren ist neu. Dies erlaubt die Herstellung von Produkten, die sowohl einen schnell einsetzenden, breiten erregerunspezifischen Schutz gegen Infektionen vermitteln als auch einen langanhaltenden, erregerspezifischen Infektionsschutz bieten.The combination of the immunostimulatory properties of PPV with the expression of foreign antigens which induce homologous and / or heterologous pathogen-specific protection is novel. This allows the production of products that provide both rapid onset, broad pathogen-specific protection against infection and provide long-lasting, pathogen-specific infection protection.
Die Familie der Vertebraten-Pockenviren (Chordopoxvirinae) wird in einzelne, eigenständige Gattungen (Genera) unterteilt. Die vorliegende Erfindung betrifft die Gattung der PPV, die sich sowohl strukturell als auch genetisch von den übrigen Pockenviren unterscheiden. Die PPV werden in drei verschiedene Spezies eingeteilt (Lit. #1):
- Parapoxvirus ovis (auch Ecthyma Contagiosum Virus, Virus der Kontagiösen Pustulardermitis oder Orf Virus genannt), das als Prototyp des Genus gilt,
- Parapoxvirus bovis 1 ( auch Bovine Papulöse Stomatitis Virus oder Stomatitis Papulosa Virus genannt) und
- Parapoxvirus bovis 2 (auch Euterpocken Virus, Paravaccinia Virus, Pseudokuhpocken Virus oder Melkerknoten Virus genannt).
- Parapoxvirus ovis (also called Ecthyma Contagiosum Virus, Contagious Pustulardermitis Virus or Orf Virus), which is considered a prototype of the genus,
- Parapoxvirus bovis 1 (also called bovine papular stomatitis virus or stomatitis papulosa virus) and
- Parapoxvirus bovis 2 (also called udderpox virus, paravaccinia virus, pseudo-chickenpox virus or milking knot virus).
Es sind auch Parapockenvirus-Vertreter beschrieben, die aus Kamelen, Rothirschen, Gemsen, Robben, Seehunden und Seelöwen isoliert wurden. Ob es sich hierbei um eigenständige Spezies innerhalb der Gattung Parapockenviren oder um Isolate der oben beschriebenen Spezies handelt, ist noch nicht endgültig geklärt.Also described are parapoxvirus representatives isolated from camels, red deer, chamois, seals, seals and sea lions. Whether it is a stand-alone species within the genus Parapox viruses or isolates of the species described above, is not yet finally resolved.
Infektionen mit PPV können lokale Erkrankungen beim Tier und beim Menschen (Zoonose-Erreger) hervorrufen. Lit. #1 gibt einen Überblick über die bislang beschriebenen Krankheitsbilder. Die Bekämpfung der Erkrankungen ist möglich durch den Einsatz von prophylaktischen Maßnahmen, wie beispielsweise Vakzinen. Die bislang erhältlichen Vakzinen, die ausschließlich auf Basis von Parapopxvirus ovis entwickelt wurden, zeigen allerdings unbefriedigende Wirksamkeit (Lit. #2).PPV infections can cause local diseases in animals and humans (zoonotic agents).
Gegenstand der Erfindung ist es, das PPV als Träger (Vektor) von fremder genetischer Information, die exprimiert wird, zu nutzen.The object of the invention is to utilize the PPV as a carrier (vector) of foreign genetic information which is expressed.
Vektoren auf Basis von Avipox-, Raccoonpox, Capripox, Swinepox oder Vaccinia Virus sind als Vektoren zur Expression von fremder genetischer Information bereits beschrieben. Die dabei gewonnenen Erkenntnisse können nicht auf PPV übertragen werden. Wie vergleichende Untersuchungen gezeigt haben, gibt es morphologische, strukturelle und genetische Unterschiede zwischen den einzelnen Gattungen der Pockenviren. So lassen sich beipielsweise die PPV mit serologischen Methoden von den anderen Gattungen der Pockenviren unterscheiden, was auf unterschiedliche Proteinmuster und damit verbunden auf unterschiedliche Erbinformationen zurückzuführen ist. Einige Vetreter der Pockenviren haben beispielsweise die Fähigkeit zur Agglutination von Erythrozyten. Diese Aktivität wird über ein Oberflächenprotein vermittelt, das sogenannte Haemagglutinin (HA). PPV haben diese Aktivität nicht.Vectors based on avipox, raccoonpox, capripox, swinepox or vaccinia virus have already been described as vectors for the expression of foreign genetic information. The insights gained can not be transferred to PPV. As comparative studies have shown, there are morphological, structural and genetic differences between the individual genera of the poxviruses. For example, the PPV can be differentiated from the other genera of the pox viruses with serological methods, which is due to different protein patterns and thus to different genetic information. For example, some representatives of the poxviruses have the ability to agglutinate erythrocytes. This activity is mediated by a surface protein, the so-called haemagglutinin (HA). PPVs do not have this activity.
Die Kenntnisse über die Organisation des Genoms von PPV beschränken sich derzeit auf die Bestimmungen der Größe des Genoms, den GC-Gehalt der Nukleinsäure, vergleichende Restriktionsenzym-Analysen, die Klonierung einzelner Genomfragmente, Sequenzanalysen von Teilbereichen und die damit verbundene, präliminäre Beschreibung von einzelnen Genen (zur Übersicht Lit. #1, Lit. #5, Lit. #6).The knowledge of the organization of the genome of PPV is currently limited to the size of the genome, the GC content of the nucleic acid, comparative restriction enzyme analyzes, the cloning of individual genome fragments, sequence analysis of subdomains and the related, preliminary description of individual genes (for
Bei Vaccinia bekannte Insertionsstellen lassen sich aufgrund ihrer fehlenden oder nicht nachgewiesenen Existenz bei PPV derzeit nicht nutzen.At Vaccinia known insertion sites can not currently be used because of their lack or undetermined existence in PPV.
So brachten z.B. die Versuche, das Gen für Thymidinkinase im Genom des PPV zu identifizieren und, wie bei Orthopockenviren als Insertionsstelle zu nutzen, keine Ergebnisse. Mazur und Mitarbeiter (Lit. #3) beschreiben zwar die Identifizierung eines Genomabschnittes von PPV, der Ähnlichkeiten mit dem Thymidinkinase-Gen von Vaccinia Virus (ein Orthopockenvirus) haben soll, eigene umfangreiche Untersuchungen konnten allerdings die Existenz eines solchen Gens bei PPV nicht bestätigen. Auch andere Autoren (Lit. #1) konnten ein Thymidinkinase-Gen bei PPV nicht finden. Bei Vaccinia Virus wird das Gen für HA als Insertionsstelle für Fremd-DNA benutzt. PPV haben diese Aktivität, wie oben beschrieben, nicht.For example, attempts to identify the gene for thymidine kinase in the genome of the PPV and to use it as an insertion site, as in orthopoxviruses, gave no results. Although Mazur and coworkers (Ref. # 3) describe the identification of a genome portion of PPV that is said to be similar to the thymidine kinase gene of vaccinia virus (an orthopox virus), extensive in-depth studies have failed to confirm the existence of such a gene in PPV. Other authors (Ref. # 1) could not find a thymidine kinase gene in PPV. In vaccinia virus, the gene for HA is used as an insertion site for foreign DNA. PPVs do not have this activity as described above.
Robinson und Lyttle erwähnten 1992 alternative Insertionstellen auf dem Genom von PPV (Lit. #1), ohne jedoch eine Beschreibung oder genaue Charakterisierung dieser Stellen zu gegeben. Auch erfolgte bis heute noch keine Beschreibung einer erfolgreichen Anwendung von PPV als Vektoren.Robinson and Lyttle mentioned alternative insertion sites on the genome of PPV in 1992 (ref. # 1), but without giving a description or exact characterization of these sites. Also, no description of a successful application of PPV as vectors has yet been made.
Bei den eigenen Untersuchungen zur Sequenzanalyse des HindIII-Fragmentes I von PPV Stamm D1701 wurde ein ORF mit Aminosäurehomologie (36,1 bis 38,3% Identität; 52,8 bis 58,6% Ähnlichkeit, GCG, Wisconsin Package 8.1, z.B. Programm Pikup) zum Vaskulären Endothelialen Wachstumsfaktor (VEGF) verschiedener Säugerspezies (z.B. Maus, Ratte, Meerschweinchen, Rind, Mensch) gefunden. Seq. ID No: 1 zeigt die Nukleotidsequenz des Gens bei D1701, Seq. ID No: 15 die Aminosäuresequenz des entsprechenden Proteins von D1701. Kürzlich wurde ein homologes Gen auch bei den PPV Stämmen NZ2 und NZ7 beschrieben (Lit. #6), dessen Funktion aber nicht bekannt ist. Ein entsprechendes Gen ist bei anderen Pockenviren, z.B. Orthopockenviren, nicht bekannt. Im weiteren Verlauf des Textes wird dieses Gen als VEGF-Gen bezeichnet.In our own investigations into the sequence analysis of the HindIII fragment I of PPV strain D1701, an ORF with amino acid homology (36.1 to 38.3% identity, 52.8 to 58.6% similarity, GCG, Wisconsin Package 8.1, eg program Pikup ) to the vascular endothelial growth factor (VEGF) of various mammalian species (eg mouse, rat, guinea pig, bovine, human). Seq. ID No: 1 shows the nucleotide sequence of the gene in D1701, Seq. ID No: 15 is the amino acid sequence of the corresponding protein of D1701. Recently, a homologous gene has also been described in the PPV strains NZ2 and NZ7 (ref. # 6), but its function is not known. A corresponding gene is present in other poxviruses, e.g. Orthopoxviruses, not known. Later in the text, this gene is called the VEGF gene.
Unsere Sequenzanalyse des HindIII-Fragmentes I von D1701 führte zur Identifizierung eines weiteren ORF, der Homologie zu Proteinkinase-Genen von Orthopockenviren hat und bei Vaccinia als F10L bekannt ist. Die Identität zum Vaccinia F10L-Gen beträgt 51%, die Ähnlichkeit 70%. Im weiteren Verlauf der Anmeldung wird dieses Gen PK-Gen genannt. Seq. ID No: 2, No: 9, No: 13 zeigt Versionen der Nukleotidsequenz des Gens bei D1701, Seq. ID No: 14 die Aminosäuresequenz des entsprechenden Proteins von D1701.Our sequence analysis of the HindIII fragment I of D1701 led to the identification of another ORF that has homology to protein kinase genes of orthopox viruses and is known as Vaccinia as F10L. The identity to the vaccinia F10L gene is 51%, the similarity 70%. In the further course of the application, this gene is called the PK gene. Seq. ID No: 2, No: 9, No: 13 shows versions of the nucleotide sequence of the gene in D1701, Seq. ID No: 14 the amino acid sequence of the corresponding protein of D1701.
Es wurde weiterhin ein ORF gefunden, der das 3'-Ende des PK-Gens und das 5'-Ende des VEGF-Gens überlappt. Homologie-Untersuchungen zeigten eine geringe Identität (28%) und geringe Ähnlichkeit (51%) zum F9L-Gen bei Vaccinia. Seq. ID No: 5, No: 10, zeigt Versionen der Nukleotidsequenz des Gens bei D1701. Im weiteren Verlauf des Textes wird dieses Gen das F9L-Gen genannt.An ORF was also found to overlap the 3 'end of the PK gene and the 5' end of the VEGF gene. Homology studies showed low identity (28%) and low similarity (51%) to the F9L gene in vaccinia. Seq. ID No: 5, No: 10, shows versions of the nucleotide sequence of the gene at D1701. Later in the text, this gene is called the F9L gene.
Innerhalb der ITR-Region wurde ein weiterer ORF gefunden, der aufgrund seiner Ähnlichkeit zu einem Gen bei PPV NZ2 (Identität 76%, Ähnlichkeit 83%) mit ORF3 bezeichnet wird. Seq. ID No: 4 zeigt die Nukleotidsequenz des Gens bei D1701. Im weiteren Verlauf des Textes wird dieses Gen mit ORF3-Gen bezeichnet.Within the ITR region, another ORF was identified, which is called ORF3 because of its similarity to a gene in PPV NZ2 (identity 76%, similarity 83%). Seq. ID No: 4 shows the nucleotide sequence of the gene at D1701. In the further course of the text, this gene is called ORF3 gene.
Gegenstand der vorliegenden Erfindung sind:
- 1. Rekombinant hergestellte PPV mit Insertionen und/oder Deletionen, enthaltend ein Fremdantigen, welches einen erregerspezifischen Schutz induzieren kann.
- 2. Rekombinant hergestellte PPV mit Insertionen und/oder Deletionen, enthaltend ein Zytokin.
- 3. Rekombinant hergestellte PPV gemäss 1
oder 2, enthaltend Insertionen und/oder Deletionen in Genomabschnitten, die nicht für die Virusvermehrung notwending sind. - 4. Rekombinant hergestellte PPV gemäss 1
oder 2, enthaltend Insertionen und/oder Deletionen in Genomabschnitten, die für die Virusvermehrung notwending sind.
- 1. Recombinantly produced PPV with insertions and / or deletions, containing a foreign antigen, which can induce a pathogen-specific protection.
- 2. Recombinantly produced PPV with insertions and / or deletions containing a cytokine.
- 3. Recombinantly produced PPV according to 1 or 2, containing insertions and / or deletions in genome sections, which are not necessary for the virus propagation.
- 4. Recombinantly produced PPV according to 1 or 2, containing insertions and / or deletions in genome sections necessary for virus replication.
Die vorstehend aufgeführten Begriffe haben folgende Bedeutung:
- Attenuierung ist ein
Prozeß bei dem die PPV durch Veränderung in ihrem Genom vermindert- oder nicht-pathogen bzw. -virulent für Tiere oder Menschen geworden sind. - Deletionen sind
fehlende DNA-Teilstücke aus dem Genom von PPV. - Deletionsplasmide sind
Plasmide, die neben der Plasmid-DNA PPV-Genomabschnitte tragen, denen Teilstücke entfernt wurden. - Zur Virusvermehrung notwendige (essentielle) Genomabschnitte sind
Teile des Gesamt-Genoms von PPV, die zur in vitro-Vermehrung von PPV, d.h. zur Bildung von infektiösen Virusnachkommen unverzichtbar sind.
Eine Störung von Genen, die für die Virusvermehrung essentiell sind, führt zu einer Unterbrechnung in der Virusvermehrung. Entfernt man beispielsweise Teile eines dieser Gene oder das gesamte Gen, bricht die Virusreplikation an einer bestimmten Stelle im Vermehrungszyklus des Virus ab. Infektionen oder Behandlungen mit solchen Mutanten führen nicht zu einer Freisetzung infektiöser Nachkommen aus dem Tier. Ersetzt man Teile eines oder ein gesamtes essentielles Gene(s) durch Fremd-DNA oder inseriert Fremd-DNA in essentielle Gene, können Vektorvakzinen konstruiert werden, die im Impfling nicht vermehrungsfähig sind und somit nicht als infektiöse Erreger ausgeschieden werden. - Zur Virusvermehrung nicht-notwendige (nicht-essentielle) Genomabschnitte sind
Teile des Gesamt-Genoms von PPV, die zur in vitro-Vermehrung von PPV, d.h. zur Bildung von infektiösen Virusnachkommen, verzichtbar sind. - Fremde DNA-Elemente (Fremd-DNA) sind
DNA-Teilstücke, z.B. Fremd-Gene oder Nukleotidsequenzen, die in dem erfindungsgemäß eingesetzten PPV ursprünglich nicht vorhanden sind.
Fremd-DNA wird aus folgenden Gründen in das PPV inseriert:- 1. zur Expression der Fremd-DNA
- 2. zur Inaktivierung von Funktionen von DNA-Teilstücken des PPV
- 3. zur Markierung des PPV.
In Abhängigkeit von diesen Gründen wird unterschiedliche Fremd-DNA inseriert. Soll gemäß (1) Fremd-DNA exprimiert werden, so wird die inserierte Fremd-DNA mindestens einen offenen Leserahmen tragen, der für ein oder mehrere gewünschte Fremdprotein(e) kodiert. Gegebenenfalls enthält die Fremd-DNA zusätzlich eigene oder fremde Regulatorsequenzen. Die Kapazität zur Aufnahme von Fremd-DNA kann durch Setzen von Deletionen im Genom des Virus vergrößert werden. Im allgemeinen liegt die Länge zwischen 1-Nukleotid und 35.000, bevorzugt zwischen 100 und etwa 15.000 Nukleotiden.
Als Beispiele seien genannt, Gene oder Genteile von Viren, wie beispielsweise- Herpesvirus suid 1,
- Equine Herpesviren
- Bovine Herpesviren
- Maul- und Klauenseuche-Virus,
- Bovines Respiratorisches Syncytial Virus,
Parainfluenzavirus 3 des Rindes,- Influenzavirus,
- Calicivirus
- Flaviviren, z.B. Bovines Virus Diarrhoe Virus oder Virus der klassischen Schweinepest
- Pasteurella spec.;
- Salmonella spec.,
- Actinobacillus spec.,
- Chlamydia spec.,
- Toxoplasma,
- Dirofilaria,
- Echinococcus.
Soll gemäß (2) Fremd-DNA inseriert werden, reicht für die Unterbrechung der Vektor-Virus-DNA-Sequenz prinzipiell die Insertion eines geeigneten Fremdnukleotids. Die maximale Länge der zur Inaktivierung inserierten Fremd-DNA richtet sich nach der Aufnahmekapazität des Vektor-Virus für Fremd-DNA. Im allgemeinen ist die Länge der Fremd-DNA zwischen 1 Nukleotid und 35.000 Nukleotiden, bevorzugt zwischen 100 und 15.000 Nukleotiden, besonders bevorzugt zwischen 3 bis 100 Nukleotiden.
Sollen gemäß (3) DNA-Sequenzen zur Markierung inseriert werden, richtet sich ihre Länge nach der Nachweismethode zur Identifizierung des markierten Virus. Im allgemeinen ist die Länge der Fremd-DNA zwischen 1 und 25.000 Nukleotiden, bevorzugt zwischen 20 Nukleotiden und 15.000 Nukleotiden, besonders bevorzugt zwischen 5 bis 100 Nukleotiden. - Genbank
ist die Gesamtheit von in vermehrungsfähigen Vektoren enthaltenen Fragmente eines Genoms. Sie wird durch Fragmentierung des Genoms und Insertion aller, einzelner oder eines Großteils der Fragmente in vermehrungsfähige Vektoren, wie z.B. Plasmide, erhalten. - Genomfragment ist
ein Teilstück eines Genoms, das isoliert vorliegen oder in einen vermehrungsfähigen Vektor inseriert sein kann. - Inaktivierung durch Insertion bedeutet,
daß die inserierte Fremd-DNA die Expression oder Funktion PPV-eigener Genom-Sequenzen verhindert. - Insertionen sind
DNA-Teilstücke, die zusätzlich in das Genom von PPV eingebaut wurden. Die Länge der DNA-Teilstücke kann je nach Grundder Insertion zwischen 1 Nukleotid und mehreren tausend Nukleotiden liegen (siehe auch Definition für "Fremd-DNA"). - Insertionsplasmide sind
Plasmide, insbesondere bakterielle Plasmide, die die zu inserierende Fremd-DNA, flankiert von DNA-Sequenzen des PPV, beinhalten. - Insertionsstellen sind
Stellen in einem Virus-Genom, die zur Aufnahme von Fremd-DNA geeignet sind. - Klonierung heißt,
daß die genomische DNA von PPV isoliert und fragmentiert wird. Die Fragmente oder eine Auswahl der Fragmente werden in gängige DNA-Vektoren (bakterielle Plasmide oder Phagenvektoren oder eukaryontische Vektoren) inseriert.
Eine Auswahl an Methoden zur Herstellung und Klonierung von DNA-Fragmenten gibt Lit. #11. Die DNA-Vektoren mit den PPV-DNA-Fragmenten als Inserts dienen z.B. der Herstellung von identischen Kopien der ursprünglich isolierten DNA-Fragmente von PPV. - Markierung durch Insertion bedeutet,
daß die inserierte Fremd-DNA die spätere Identifizierung des veränderten PPV ermöglicht. - Unter ORF (Open Reading Frame) versteht man eine Abfolge von Nukleotiden auf DNA-Ebene, die die Aminosäuresequenz eines potentiellen Proteins definiert. Sie besteht aus einer durch die Größe des von ihr definierten Proteins bestimmten Anzahl von Nukleotid-Tripletts, die 5'-seitig durch ein Startcodon (ATG) und 3'-seitig durch ein Stopcodon (TAG, TGA, TAA) begrenzt wird.
- Regulatorsequenzen sind
DNA-Sequenzen, die die Expression von Genen beeinflussen. Solche sind bekannt aus Lit. #15.
Bevorzugt sei genannt der VEGF-Promotor, wie er in Sequenz Protokoll ID No: 6 beschrieben ist. - Rekombinante PPV sind
PPV mit Insertionen und/oder Deletionen in ihrem Genom. Die Insertionen und Deletionen wurden hierbei über molekularbiologische Methoden hergestellt. - Repetitive (DNA)-Sequenzen sind
identische Nukleotid-Sequenzen, die im PPV-Genom direkt nacheinander oder verstreut an unterschiedlichen Stellen vorkommen. - Vektor-Virus ist
ein PPV, das zur Insertion von Fremd-DNA geeignet ist und die inserierte Fremd-DNA in seinem Genom in infizierte Zellen oder Organismen transportieren kann und gegebenenfalls die Expression der Fremd-DNA ermöglicht.
- Attenuation is one
Process in which the PPV have become degraded or non-pathogenic or virulent to animals or humans by alteration in their genome. - Deletions are
missing DNA fragments from the genome of PPV. - Deletion plasmids are
Plasmids that carry PPV genome sections in addition to the plasmid DNA that have been removed. - Necessary for virus replication (essential) genome sections are
Portions of the total genome of PPV indispensable for the in vitro propagation of PPV, that is, for the generation of infectious progeny virus.
A disruption of genes that are essential for virus replication leads to an interruption in the virus multiplication. If, for example, parts of one of these genes or the entire gene are removed, virus replication breaks down at a certain point in the reproductive cycle of the virus. Infections or treatments with such mutants do not result in the release of infectious offspring from the animal. Replacing parts of or an entire essential gene (s) by foreign DNA or inserting foreign DNA into essential genes, vector vaccines can be constructed that are not replicable in the vaccine and thus are not excreted as infectious agents. - For virus replication, non-essential (nonessential) genome segments are
Parts of the total genome of PPV, which are dispensable for the in vitro propagation of PPV, ie for the formation of infectious virus progeny. - Foreign DNA elements (foreign DNA) are
DNA fragments, eg foreign genes or nucleotide sequences, which are not originally present in the PPV used according to the invention.
Foreign DNA is inserted into the PPV for the following reasons:- 1. to the expression of the foreign DNA
- 2. Inactivation of functions of DNA fragments of the PPV
- 3. to mark the PPV.
Depending on these reasons, different foreign DNA is inserted. If foreign DNA is to be expressed according to (1), the inserted foreign DNA will carry at least one open reading frame which codes for one or more desired foreign protein (s). Optionally, the foreign DNA additionally contains its own or foreign regulatory sequences. The capacity for uptake of foreign DNA can be increased by setting deletions in the genome of the virus. In general, the length is between 1 nucleotide and 35,000, preferably between 100 and about 15,000 nucleotides.
Examples include genes or genetic parts of viruses, such as- Herpesvirus suid 1,
- Equine herpesviruses
- Bovine herpesviruses
- Foot-and-mouth disease virus,
- Bovine Respiratory Syncytial Virus,
- Parainfluenza
virus 3 of bovine, - Influenza virus,
- calicivirus
- Flaviviruses, eg Bovine virus diarrhea virus or classical swine fever virus
- Pasteurella spec .;
- Salmonella spec.,
- Actinobacillus spec.,
- Chlamydia spec.,
- Toxoplasma,
- Dirofilaria,
- Echinococcus.
If according to (2) foreign DNA is inserted, the insertion of a suitable foreign nucleotide is sufficient in principle for the interruption of the vector virus DNA sequence. The maximum length of the foreign DNA inserted for inactivation depends on the uptake capacity of the vector virus for foreign DNA. In general, the length of the foreign DNA is between 1 nucleotide and 35,000 nucleotides, preferably between 100 and 15,000 nucleotides, more preferably between 3 to 100 nucleotides.
If, according to (3), DNA sequences are to be inserted for labeling, their length depends on the detection method for identification of the labeled virus. In general, the length of the foreign DNA is between 1 and 25,000 nucleotides, preferably between 20 nucleotides and 15,000 nucleotides, more preferably between 5 to 100 nucleotides. - Genbank
is the set of fragments of a genome contained in replicable vectors. It is obtained by fragmenting the genome and inserting all, a single or a majority of the fragments into replicable vectors, such as plasmids. - Genome fragment is
a portion of a genome that may be isolated or inserted into a replicable vector. - Inactivation by insertion means
that the inserted foreign DNA prevents the expression or function of PPV's own genome sequences. - Insertions are
DNA fragments additionally incorporated into the genome of PPV. The length of the DNA fragments can be between 1 nucleotide and several thousand nucleotides, depending on the reason for the insertion (see also definition for "foreign DNA"). - Insertion plasmids are
Plasmids, in particular bacterial plasmids, which contain the foreign DNA to be inserted, flanked by DNA sequences of the PPV. - Insertion sites are
Sites in a virus genome that are suitable for receiving foreign DNA. - Cloning means
that the genomic DNA of PPV is isolated and fragmented. The fragments or a selection of the fragments are inserted into common DNA vectors (bacterial plasmids or phage vectors or eukaryotic vectors).
For a selection of methods for preparing and cloning DNA fragments, see Ref. # 11. The DNA vectors with the PPV DNA fragments as inserts serve, for example, to produce identical copies of the originally isolated DNA fragments of PPV. - Marking by insertion means
that the inserted foreign DNA allows later identification of the altered PPV. - ORF (Open Reading Frame) is a sequence of DNA-level nucleotides that defines the amino acid sequence of a potential protein. It consists of a number of nucleotide triplets determined by the size of the protein defined by it, which is bounded on the 5 'side by a start codon (ATG) and on the 3' side by a stop codon (TAG, TGA, TAA).
- Regulator sequences are
DNA sequences that influence the expression of genes. Such are known fromRef. # 15.
Preference should be given to the VEGF promoter as described in Sequence Protocol ID No: 6. - Recombinant PPV are
PPV with insertions and / or deletions in their genome. The insertions and deletions were produced by molecular biological methods. - Repetitive (DNA) sequences are
identical nucleotide sequences that occur directly in the PPV genome or scattered at different sites. - Vector virus is
a PPV which is suitable for insertion of foreign DNA and which can transport the inserted foreign DNA in its genome into infected cells or organisms and optionally allows expression of the foreign DNA.
Die Herstellung der erfindungsgemäßen PPV erfolgt wie folgt beschrieben:
- 1.
- Auswahl eines geeigneten PPV-Stammes
- 2.
- Identifizierung von Genomabschnitten mit Insertionsstellen in dem Genom des PPV
- 2.a
- Identifizierung von PPV-Genomabschnitten mit Insertionsstellen in Genen, die nicht-essentiell für die Virusvermehrung sind,
- 2.b
- Identifizierung von PPV Genomabschnitten mit Insertionsstellen in Genen, die essentiell für die Virusvermehrung sind,
- 2.c
- Identifizierung von Genomabschnitten mit Insertionsstellen in Bereichen außerhalb von Genen in dem Genom des PPV und/oder in Genduplikationen
- 2.d
- Weitere Methoden zur Identifizierung von Genomabschnitten mit Insertionsstellen
- 2.e
- Anforderungen an eine Insertionsstelle
- 2.1
- Identifizierung von Insertionsstellen
- 2.1.1
- Reinigung des Genoms von PPV
- 2.1.2
- Klonierung der Genomfragmente, Etablierung einer Genbank
- 2.1.3
- Sequenzierung zur Identifizierung von Genen oder Genomabschnitten außerhalb von Genen
- 2.1.4
- Auswahl der Klone mit PPV-Genomfragmenten zur Weiterverarbeitung
- 2.2
- ITR-Region, VEGF-, PK-Gen, Gen kodierend für das 10 kDa-Protein und der Bereich zwischen dem PK- und dem HDIR-Gen als Insertionsstellen
- 2.2.1
- Klonierung des VEGF-Gens
- 2.2.2
- Klonierung des Proteinkinase-Gens
- 2.2.3
- Klonierung des Gen-Bereichs der für das 10kDa-Protein kodiert
- 2.2.4
- Klonierung der ITR-Region (Inverted-Terminal-Repeat-Region) bzw. des Genomabschnittes, der zwischen dem PK-Gen und dem HD1R-Gen liegt
- 3.
- Konstruktion von Insertionsplasmiden oder von Deletionsplasmiden, die die zu inserierende Fremd-DNA enthalten,
- 3.1
- Identifizierung oder Herstellung von nur einmal vorkommenden, d.h. singulären Restriktionsenzymerkennungsstellen ("unique restriction sites") in den klonierten Genomfragmenten und Insertion von Fremd-DNA
- 3.2
- Deletion von Genomsequenzen in den klonierten Genomfragmenten und Insertion von Fremd-DNA
- 3.3
- Eine Kombination von #3.1 und #3.2
- 4.
- Konstruktion eines rekombinanten PPV
- 1.
- Selection of a suitable PPV strain
- Second
- Identification of genomic regions with insertion sites in the genome of the PPV
- 2.a
- Identification of PPV genomic regions with insertion sites in genes that are non-essential for virus replication,
- 2 B
- Identification of PPV genome segments with insertion sites in genes essential for virus replication
- 2.c
- Identification of genomic regions with insertion sites in out-of-gene regions in the genome of the PPV and / or in gene duplications
- 2.d
- Further methods for the identification of genome sections with insertion sites
- 2.e
- Requirements for an insertion site
- 2.1
- Identification of insertion sites
- 2.1.1
- Purification of the genome of PPV
- 2.1.2
- Cloning of genomic fragments, establishment of a gene bank
- 2.1.3
- Sequencing for the identification of genes or genome segments outside of genes
- 2.1.4
- Selection of clones with PPV genome fragments for further processing
- 2.2
- ITR region, VEGF, PK gene, gene coding for the 10 kDa protein and the region between the PK and the HDIR gene as insertion sites
- 2.2.1
- Cloning of the VEGF gene
- 2.2.2
- Cloning of the protein kinase gene
- 2.2.3
- Cloning the gene region encoding the 10kDa protein
- 2.2.4
- Cloning of the ITR region (inverted terminal repeat region) or of the genome segment which lies between the PK gene and the HD1R gene
- Third
- Construction of insertion plasmids or of deletion plasmids containing the foreign DNA to be inserted,
- 3.1
- Identification or production of unique, ie unique restriction restriction recognition sites in the cloned genomic fragments and insertion of foreign DNA
- 3.2
- Deletion of genome sequences in the cloned genomic fragments and insertion of foreign DNA
- 3.3
- A combination of # 3.1 and # 3.2
- 4th
- Construction of a recombinant PPV
Grundsätzlich sind alle PPV-Spezies für die Durchführung der vorliegenden Erfindung geeignet. Bevorzugt werden Virusstämme, die sich zu Titern > 105 PFU (Plaque Forming Unit)/ml in einer Gewebekultur vermehren lassen und aus dem Medium der infizierten Zellen als extrazelluläres, infektiöses Virus rein darstellen lassen. Bevorzugt genannt sei vom Genus der PPV die Spezies PPV ovis (Orf Viren).Basically, all PPV species are suitable for carrying out the present invention. Preference is given to virus strains which can be multiplied to titer> 10 5 PFU (plaque forming unit) / ml in a tissue culture and can be purified from the medium of the infected cells as an extracellular, infectious virus. Preference is given to the genus of PPV the species PPV ovis (Orf viruses).
Als besonders bevorzugt genannt sei der PPV-ovis-Stamm D1701, hinterlegt nach dem Budapester Vertrag am 28.04.1988 bei Institut Pasteur, C.N.C.M. unter der Reg.Nr. CNCM I-751, sowie seine Varianten und Mutanten.Particular preference is given to the PPV ovis strain D1701, deposited under the Budapest Treaty on 28.04.1988 at Institut Pasteur, CNCM under Reg.No. CNCM I-751, as well as its variants and mutants.
Die Vermehrung der Viren erfolgt in üblicher Weise in Gewebekulturen animaler Zellen wie Säugetierzellen, z.B. in Schafzellen, Rinderzellen, bevorzugt in Rinderzellen wie der permanenten Rindernierenzelle BK-K1-3A (oder deren Abkömmlingen) oder Affenzellen wie den permanenten Affennierenzellen MA104 oder Vero (oder deren Abkömmlingen).The multiplication of the viruses takes place in a customary manner in tissue cultures of animal cells, such as mammalian cells, for example in ovine cells, bovine cells, preferably in bovine cells such as the permanent bovine kidney cell BK-K1-3A (or its progeny) or monkey cells such as the permanent monkey kidney cells MA104 or Vero (or their progeny).
Die Vermehrung erfolgt in an sich bekannter Weise in stationären, Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden oder in Suspensionskulturen.Propagation is carried out in a manner known per se in stationary, roller or carrier cultures in the form of closed cell aggregates or in suspension cultures.
Die zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zu optimalen Zelldichte vermehrt. Die Zellen werden mit Virusverdünnungen infiziert, die einer MOI (= multiplicity of infection, entspricht infektiöse Viruspartikel pro Zelle) entsprechen.The cells and cell turfs used for the multiplication of the viruses are increased in the usual way almost to confluence or up to optimal cell density. The cells are infected with virus dilutions that correspond to an MOI (= multiplicity of infection, corresponding to infectious virus particles per cell).
Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einer Konzentration von 1-30 Vol.-%, vorzugsweise 1-10 Vol.-% zugegeben.The multiplication of the viruses takes place with or without the addition of animal serums. In the event that serum is used, this is added to the propagation medium in a concentration of 1-30 vol .-%, preferably 1-10 vol .-%.
Infektion und Virusvermehrung erfolgt bei Temperaturen zwischen Raumtemperatur und 40° C, bevorzugt zwischen 32 und 39° C, besonders bevorzugt bei 37° C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zellen. Bei der Virusernte kann noch zellgebundenes Virus mechanisch oder durch Ultraschall oder milde enzymatische Proteolyse z.B. mit Trypsin zusätzlich freigesetzt werden.Infection and virus multiplication takes place at temperatures between room temperature and 40 ° C, preferably between 32 and 39 ° C, more preferably at 37 ° C for several days, preferably until complete destruction of the infected cells. In the virus harvest, cell-bound virus can be mechanically or by ultrasound or mild enzymatic proteolysis e.g. be additionally released with trypsin.
Das virushaltige Medium der infizierten Zellen kann dann weiter aufgearbeitet werden, z.B. durch Entfernung der Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,2-0,45 µm und/oder niedertourige Zentrifugation.The virus-containing medium of the infected cells can then be further worked up, e.g. by removing the cell debris by filtration with pore sizes of e.g. 0.2-0.45 μm and / or low speed centrifugation.
Filtrat oder Zentrifugationsüberstand können zur Virusanreicherung und - reinigung verwendet werden. Dazu werden Filtrat oder Überstand einer hochtourigen Zentrifugation bis zur Sedimentation der Viruspartikel unterworfen. Gegebenenfalls können weitere Reinigungsschritte durch z.B. Zentrifugation in einem Dichtegradienten angeschlossen werden.Filtrate or centrifugation supernatant can be used for virus replenishment and purification. For this purpose, the filtrate or supernatant of a high-speed centrifugation are subjected to sedimentation of the virus particles. Optionally, further purification steps can be connected by, for example, centrifugation in a density gradient.
Verschiedene Bereiche des Genoms des PPV können als Insertionsstellen zur Insertion von Fremd-DNA dienen. Fremd-DNA kann inseriert werden,
- a. in Gene, die nicht-essentiell für die Virusvermehrung in vitro und/oder in vivo sind, .
- b. in Gene, die essentiell für die Virusvermehrung sind und/oder
- c. in Bereichen ohne Genfunktion.
- a. in genes that are non-essential for viral replication in vitro and / or in vivo,.
- b. in genes that are essential for virus replication and / or
- c. in areas without gene function.
- i. Virale Gene, die nicht-essentiell für die Virusvermehrung sind, werden beispielsweise durch vergleichende Untersuchungen mit Vertretern verschiedener PPV-Spezies gefunden. Gene, die in einem oder mehreren Isolaten oder Stämmen einer PPV-Spezies nicht vorkommen, in anderen Isolaten oder Stämmen aber gefunden werden, sind potentiell nicht-essentiell.i. Viral genes that are non-essential for virus propagation are found, for example, by comparative studies with representatives of various PPV species. Genes that are not found in one or more isolates or strains of a PPV species but are found in other isolates or strains are potentially non-essential.
- ii. Gene, die nicht-essentiell für die Virusvermehrung sind, können auch auf einem alternativen Weg identifiziert werden. Nach der Sequenzierung von Genomfragmenten von PPV, die beispielsweise als klonierte Fragmente vorliegen können, werden diese DNA-Sequenzen auf mögliche "Offene Leserahmen" ("Open Reading Frames", ORF) untersucht. Findet man ein ORF, wird dessen Funktion als Gen über den Nachweis der Transkription und/oder Translation verifiziert. Um festzustellen, ob das gefundene Gen nicht-essentiell für die Virusvermehrung ist, wird das Gen durch molekulargenetische Methoden aus dem Genom des PPV entfernt, partiell zerstört oder durch eingeführte Mutation(en) unterbrochen, und das resultierende Virus auf Vermehrungsfähigkeit untersucht. Kann das Virus auch ohne die Existenz des manipulierten Genes replizieren, handelt es sich hierbei um ein nicht-essentielles Gen.ii. Genes that are non-essential for virus replication can also be identified by an alternative route. Following sequencing of genomic fragments of PPV that may be present, for example, as cloned fragments, these DNA sequences are screened for possible "open reading frames" (ORFs). If an ORF is found, its function as a gene is verified via the detection of transcription and / or translation. To determine if the gene found is non-essential for virus replication, the gene is removed from the genome of the PPV by molecular genetic methods, partially disrupted or disrupted by introduced mutation (s), and the resulting virus assayed for replicative capacity. If the virus can also replicate without the existence of the manipulated gene, this is a non-essential gene.
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i. Als Beispiel für ein nicht-essentielles Gen von PPV sei hier das VEGF-Gen des PPV ovis genannt, das als Inertionsstelle für Fremd-DNA dient. Dieses Gen kommt in den untersuchten Stämmen von Parapoxvirus ovis (NZ-2, NZ-7 und D1701 vor (Lit. #6)). Bei einigen PPV-Stämmen, beispielsweise Vertreter von PPV bovis 1 ist dieses VEGF-Gen nicht nachgewiesen. Die Identifizierung des Bereiches mit dem VEGF-Gen auf dem Genom von PPV kann mit Hilfe der im Sequenz Protokoll ID No: 1 gezeigten DNA-Sequenz erfolgen. Über die üblichen Methoden der Molekularbiologie kann das Gen mittels Hybridisierungsexperimenten, Genom-Sequenzanalysen und/oder Polymerase-Ketten-Reaktionen auf dem Genom eines PPV gefunden werden.i. An example of a non-essential gene of PPV is the VEGF gene of the PPV ovis, which acts as an inducible site for foreign DNA. This gene is found in the strains studied by Parapoxvirus ovis (NZ-2, NZ-7 and D1701 (Ref. # 6)). In some PPV strains, for example, members of
PPV bovis 1, this VEGF gene is not detected. The identification of the region with the VEGF gene on the genome of PPV can be carried out using the DNA sequence shown in Sequence Protocol ID No: 1. By conventional molecular biology techniques, the gene can be found by hybridization experiments, genome sequence analyzes, and / or polymerase chain reactions on the genome of a PPV. - ii. Als weiteres Beispiel für ein potentiell nicht-essentielles Gen von PPV sei das Gen für das 10kDa-Protein von PPV genannt. Dieses Gen kommt in Stämmen von Parapoxvirus ovis (NZ-2, NZ-7 und D1701) vor. Die Identifizierung des Bereiches mit dem Gen für das 10kDa-Protein auf dem Genom von PPV kann mit üblichen Methoden der Molekularbiologie wie beispielsweise mit Hilfe der Polymerase-Ketten-Reaktionen (PCR) auf dem Genom eines PPV gefunden werden. Lit. #8 zeigt die DNA-Sequenz des 10kDa-Protein-Gens. Die für eine PCR einsetzbaren Primer sind beispielsweise in Lit. #8 genannt. Die DNA-Sequenz des 10kDa-spezifischen PCR-Produktes von D1701 zeigt Sequenzprotokoll ID No: 11.ii. Another example of a potentially non-essential gene of PPV is the gene for the 10kDa protein of PPV. This gene is found in strains of Parapoxvirus ovis (NZ-2, NZ-7 and D1701). The identification of the region for the 10kDa protein gene on the genome of PPV can be found on the genome of a PPV using standard molecular biology techniques, such as polymerase chain reaction (PCR). Ref. # 8 shows the DNA sequence of the 10kDa protein gene. The primers which can be used for a PCR are mentioned, for example, in reference # 8. The DNA sequence of the 10kDa-specific PCR product of D1701 shows Sequence Listing ID No: 11.
Für die Insertion von Fremd-DNA kann das nicht-essentielle Gen in Teilen oder vollständig aus dem Genom des PPV entfernt werden. Es ist aber auch möglich, Fremd-DNA in das nicht-essentielle Gen zu inserieren, ohne daß Bereiche des PPV-Gens entfernt werden. Als Insertionsstellen können beispielsweise Restriktionsenzym-Erkennungsstellen dienen.For the insertion of foreign DNA, the non-essential gene may be partially or completely deleted from the genome of the PPV. However, it is also possible to insert foreign DNA into the non-essential gene without removing portions of the PPV gene. For example, restriction enzyme recognition sites can serve as insertion sites.
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i. Die Identifizierung essentieller Gene kann durch Sequenzierung von viralen Genomfragmenten die beispielsweise als klonierte Fragmente vorliegen, und Identifizierung möglicher ORF erfolgen.
Findet man einen ORF, wird dessen Funktion als Gen über den Nachweis der Transkription und/oder Translation verifiziert. Um festzustellen, ob das gefundene Gen essentiell für die Virusvermehrung ist, wird das Gen durch molekulargenetische Methoden im Genom des PPV zerstört, beispielsweise durch Entfernung von Teilen oder des gesamten Gens oder durch Insertion von Fremd-DNA, und das resultierende Virus auf Vermehrungsfähigkeit untersucht. Kann die erhaltene Virusmutante nicht replizieren, handelt es sich hierbei mit großer Wahrscheinlichkeit um ein essentielles Gen. i. The identification of essential genes can be accomplished by sequencing viral genomic fragments present, for example, as cloned fragments, and identifying possible ORFs.
If an ORF is found, its function as a gene is verified via the detection of transcription and / or translation. To determine if the gene found is essential for virus propagation, the gene is disrupted by molecular genetic methods in the genome of the PPV, for example, by removal of parts or the whole gene, or by insertion of foreign DNA, and the resulting virus is tested for proliferation. If the viral mutant obtained can not replicate, it is likely to be an essential gene.
Beweisend ist das ausschließliche Wachstum der Virusmutante in komplementierenden Zellinien.Proof is the exclusive growth of the virus mutant in complementing cell lines.
Als Beispiel sei das Proteinkinasegen (PK-Gen) von PPV D 1701 genannt. Das PK-Gen wird spät im Vermehrungszyklus des Virus exprimiert. Die Identifizierung des PK-Gens auf dem Genom von PPV kann mit Hilfe der im Sequenz Protokoll ID No: 2, No: 9 oder No: 13 gezeigten Versionen der DNA-Sequenz erfolgen. Über die üblichen Methoden der Molekularbiologie kann der Bereich mit dem Gen beispielsweise durch Hybridisierungsexperimente, Genom-Sequenzanalysen und/oder Polymerase-Ketten-Reaktionen auf dem Genom eines PPV gefunden werden.As an example, the protein kinase gene (PK gene) of PPV D 1701 called. The PK gene is expressed late in the reproductive cycle of the virus. The identification of the PK gene on the genome of PPV can be done using the versions of the DNA sequence shown in Sequence Listing ID No: 2, No: 9 or No: 13. By the usual methods of molecular biology, the region of the gene can be found, for example, by hybridization experiments, genome sequence analyzes and / or polymerase chain reactions on the genome of a PPV.
Für die Insertion von Fremd-DNA kann das essentielle Gen in Teilen oder vollständig aus dem Genom des PPV entfernt werden. Es ist aber auch möglich, die Fremd-DNA in das essentielle Gen zu inserieren, ohne daß Bereiche aus dem PPV-Gen entfernt werden.For the insertion of foreign DNA, the essential gene can be partially or completely removed from the genome of the PPV. However, it is also possible to insert the foreign DNA into the essential gene without removing areas from the PPV gene.
Genomabschnitte, die nicht für funktionelle Genprodukte kodieren und keine essentiellen regulatorischen Funktionen besitzen (sog. intergenische Abschnitte), sind prinzipiell als Insertionsstellen für Fremd-DNA geeignet. Besonders geeignet sind Bereiche mit repetitiven Sequenzen, da Veränderungen in Teilbereichen durch verbleibende Sequenzrepetitionen kompensiert werden können. Hierunter fallen auch Gene, die in zwei- oder mehrfacher Kopie vorkommen, sogenannte Genduplikationen.Genome segments which do not code for functional gene products and have no essential regulatory functions (so-called intergenic sections) are in principle suitable as insertion sites for foreign DNA. Particularly suitable are areas with repetitive sequences, since changes in partial areas can be compensated by remaining Sequenzrepetitionen. This includes genes that occur in two or more copies, so-called gene duplications.
Gene in der ITR-Region oder in duplizierten Abschnitten des PPV-Genoms existieren in zwei Kopien im Virusgenom. Nach Entfernen oder Veränderung einer Kopie eines solchen Gens und Insertion von Fremd-DNA können stabile PPV Rekombinanten erhalten werden, auch wenn das geänderte Gen für die Virusvermehrung wichtig sein sollte. Eine zweite, ungeänderte Genkopie könnte für die Genfunktion ausreichen.
- i. Die Identifizierung von Genomsequenzen, die nicht für Genprodukte kodieren, erfolgt über Sequenzanalysen des Genoms der PPV. Genombereiche, die weder nach Sequenzanalyse einen ORF noch virusspezifische Transkription zeigen und keine regulatorische Funktion aufweisen, stellen potentielle Insertionsstellen dar. Besonders die Schnittstellen für Restriktionsenzyme in diesen Bereichen repräsentieren potentielle Insertionsstellen. Zur Überprüfung, ob eine geeignete Insertionsstelle vorliegt, wird über bekannte molekularbiologische Methoden Fremd-DNA in die potentielle Insertionsstelle inseriert und die resultierende Virusmutante auf Lebensfähigkeit untersucht. Wenn die Virusmutante, die in der möglichen Insertionstelle Fremd-DNA trägt, vermehrungsfähig ist, handelt es sich bei der untersuchten Stelle um eine geeignete Insertionstelle.
- ii. Die Identifizierung von repetitiven Sequenzen sowie Genduplikationen erfolgt beispielsweise über DNA-Hybridisierungsexperimente und/oder über Sequenzanalysen. Bei den Hybridisierungsexperimenten werden klonierte oder isolierte Genomfragmente eines PPV als Sonden für Hybridisierungen mit PPV-DNA-Fragmenten eingesetzt. Die Genomfragmente des PPV, die mit mehr als einem Fragment des Gesamtgenoms des PPV hybridisieren, enthalten eine oder mehrere repetitive Sequenzen. Um die repetitive Sequenz oder duplizierte Genombereiche auf dem Genom-Fragment genau zu lokalisieren, wird die Nukleotidsequenz bestimmt. Um festzustellen, ob eine potentielle Insertionsstelle eine geeignete Insertionsstelle im Gesamtgenom des PPV ist, muß Fremd-DNA in eine repetitive Sequenz oder in eine Kopie der Genduplikation inseriert werden und das PPV-Genom-Fragment mit dem Insert in das Virusgenom eingebaut werden. Anschließend wird das Fremd-DNA enthaltende rekombinante Virus auf Vermehrungsfähigkeit geprüft. Vermehrt sich das rekombinante Virus, ist die identifizierte Erkennungsstelle als Insertionsstelle geeignet.
- i. The identification of genome sequences which do not code for gene products is carried out via sequence analyzes of the genome of the PPV. Genome regions which show neither ORF nor virus-specific transcription after sequence analysis and have no regulatory function represent potential insertion sites. In particular, the restriction enzyme sites in these areas represent potential insertion sites. To check whether a suitable insertion site is present, foreign DNA is inserted into the potential insertion site via known molecular-biological methods and the resulting virus mutant is examined for viability. If the virus mutant carrying foreign DNA in the possible insertion site is capable of reproduction, the site of interest is an appropriate insertion site.
- ii. The identification of repetitive sequences as well as gene duplications takes place, for example, via DNA hybridization experiments and / or via sequence analyzes. In hybridization experiments, cloned or isolated genomic fragments of a PPV are used as probes for hybridization with PPV DNA fragments. The genomic fragments of the PPV that hybridize to more than one fragment of the total genome of the PPV contain one or more repetitive sequences. To the repetitive sequence or duplicated To accurately locate genomic regions on the genome fragment, the nucleotide sequence is determined. To determine if a potential insertion site is a suitable insertion site in the overall genome of the PPV, foreign DNA must be inserted into a repetitive sequence or into a copy of the gene duplication and the PPV genome fragment with the insert inserted into the viral genome. Subsequently, the foreign DNA-containing recombinant virus is tested for proliferative capacity. If the recombinant virus proliferates, the identified recognition site is suitable as an insertion site.
- i. Als Beispiele für intergenische Bereiche seien der Genomabschnitt zwischen dem Gen für die Proteinkinase und dem HDIR-Gen (Sequenz Protokoll ID No: 7) genannt.i. Examples of intergenic regions include the genome section between the gene for the protein kinase and the HDIR gene (sequence protocol ID No: 7).
- ii. Als Beispiel für eine repetitive Sequenz sei die ITR-Region genannt (Sequenz Protokoll ID No: 4).ii. An example of a repetitive sequence is called the ITR region (Sequence Protocol ID No: 4).
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iii. Als Beispiel einer Genduplikation im PPV-Stamm D1701 sei das potentielle Gen "ORF 3" (in der ITR-Region) sowie das VEGF-Gen genannt. Hybridisierungsstudien bewiesen, daß im vorliegenden Stamm D1701 ein Bereich, der das VEGF-Gen beinhaltet, dupliziert und an das andere Ende des Virusgenoms transloziert wurde, so daß zwei VEGF-Genkopien vorliegen.
Anhand der Sequenzen im Sequenz Protokoll ID No: 4 (ITR-Sequenz mit Gen "ORF3") und ID No: 7 (Bereich zwischen PK-und HDIR-Gen) können die entsprechenden Genombereiche bei anderen PPV mit üblichen Methoden der Molekularbiologie, beispielsweise Hybridisierungsexperimenten, Genom-Sequenzanalysen und/oder Polymerase-Ketten-Reaktionen gefunden werden. iii. As an example of a gene duplication in the PPV strain D1701, the potential gene "ORF 3" (in the ITR region) and the VEGF gene may be mentioned. Hybridization studies demonstrated that in the present strain D1701, an area containing the VEGF gene was duplicated and translocated to the other end of the viral genome so that there are two copies of VEGF gene.
Based on the sequences in sequence protocol ID No: 4 (ITR sequence with gene "ORF3") and ID No: 7 (region between PK and HDIR gene), the corresponding genome regions in other PPVs can be analyzed by conventional methods of molecular biology, for example hybridization experiments , Genome sequence analyzes and / or polymerase chain reactions can be found.
Generell werden mögliche Insertionsstellen auf dem Genom von PPV auch über Modifikationen der viralen Genomsequenzen gefunden. Genomstellen, an denen Nukleotid-Austausche, Deletionen und/oder Insertionen oder Kombinationen hieraus, die Virusvermehrung nicht blockieren, stellen mögliche Insertionsstellen dar. Zur Überprüfung, ob eine potentielle Insertionsstelle eine geeignete Insertionsstelle darstellt, wird über bekannte molekularbiologische Methoden Fremd-DNA in die potentielle Insertionsstelle inseriert und die resultierende Virusmutante auf Lebensfähigkeit untersucht. Wenn die Virusrekombinante vermehrungsfähig ist, handelt es sich bei der untersuchten Stelle um eine geeignete Insertionstelle.In general, possible insertion sites on the genome of PPV are also found via modifications of the viral genome sequences. Genomic sites where nucleotide exchanges, deletions and / or insertions or combinations thereof do not block viral replication represent potential sites of insertion. To test whether a potential site of insertion is a suitable insertion site, foreign DNA is introduced into the potential by known molecular biology techniques Insertion site inserted and examined the resulting virus mutant for viability. If the virus recombinant is capable of replication, the site of interest is an appropriate insertion site.
Für die molekulargenetische Klonierung von Insertionsstellen von PPV wird zunächst das Genom der PPV gereinigt. Ausgehend von dem gemäß 1 (oben) hergestellten Virus wird das Genom isoliert und gereinigt. Die Extraktion von nativer viraler DNA erfolgt bevorzugt über die Behandlung der gereinigten Virionen mit wäßrigen Lösungen von Detergentien und Proteasen.For the molecular genetic cloning of insertion sites of PPV, the genome of the PPV is first purified. Starting from the virus prepared according to 1 (above), the genome is isolated and purified. The extraction of native viral DNA is preferably carried out by treating the purified virions with aqueous solutions of detergents and proteases.
Als Detergentien seien genannt anionische, kationische, amphotere, nichtionische Detergentien. Bevorzugt werden ionische Detergentien eingesetzt. Besonders bevorzugt werden Natriumdodecylsulfat, Natriumlaurylsulfat.Detergents include anionic, cationic, amphoteric, nonionic detergents. Preferably, ionic detergents are used. Particularly preferred are sodium dodecyl sulfate, sodium lauryl sulfate.
Als Proteasen seien genannt alle Proteasen, die in Gegenwart von Detergens arbeiten, wie z.B. Proteinase K und Pronase. Bevorzugt sei genannt Proteinase K.As proteases may be mentioned all proteases which operate in the presence of detergent, e.g. Proteinase K and pronase. Preference is given to proteinase K.
Detergentien werden in Konzentrationen von 0,1-10 Vol.-% eingesetzt, bevorzugt sind 0,5-3 Vol.-%.Detergents are used in concentrations of 0.1-10 vol .-%, preferably 0.5-3 vol .-%.
Proteasen werden in Konzentrationen von 0,01-10 mg/ml Viruslysat eingesetzt, bevorzugt sind 0,05-0,5 mg/ml Viruslysat.Proteases are used in concentrations of 0.01-10 mg / ml virus lysate, preferably 0.05-0.5 mg / ml virus lysate.
Es wird bevorzugt in wäßriger gepufferter Lösung in Gegenwart von DNase-Inhibitoren gearbeitet. Als Puffersubstanzen seien genannt: Salze schwacher Säuren mit starken Basen wie z.B. Tris(hydroxymethylaminomethan), Salze starker Säuren mit schwachen Basen wie z.B. primäre Phosphate oder Gemische derselben.It is preferred to work in aqueous buffered solution in the presence of DNase inhibitors. As buffer substances may be mentioned: salts of weak acids with strong bases such as e.g. Tris (hydroxymethylaminomethane), salts of strong acids with weak bases, e.g. primary phosphates or mixtures thereof.
Bevorzugt sei das folgende Puffersystem genannt: Tris(hydroxymethylaminomethan).The following buffer system may preferably be mentioned: tris (hydroxymethylaminomethane).
Die Puffersubstanzen oder Puffersysteme werden in Konzentrationen eingesetzt, die pH-Werte gewährleisten, bei denen die DNA nicht denaturiert. Bevorzugt sind pH-Werte von 5-9, besonders bevorzugt 6-8,5 , ganz besonders bevorzugt 7-8, insbesondere sei Arbeiten im neutralen Bereich genannt.The buffer substances or buffer systems are used in concentrations which ensure pH values at which the DNA does not denature. Preference is given to pH values of 5-9, particularly preferably 6-8.5, completely particularly preferred 7-8, in particular work in the neutral range is called.
DNase-Inhibitoren sind z.B. Ethylendiamintetraessigsäure in Konzentrationen von 0,1-10 mM (Millimol), bevorzugt ist ca. 1 mM.DNase inhibitors are e.g. Ethylenediaminetetraacetic acid in concentrations of 0.1-10 mM (millimoles), preferably about 1 mM.
Anschließend werden die lipophilen Bestandteile des Viruslysats extrahiert. Als Extraktionsmittel dienen Lösungsmittel wie Phenol, Chloroform, Isoamylalkohol oder deren Gemische. Bevorzugt wird zunächst ein Gemisch aus Phenol und Chloroform/Isoamylalkohol eingesetzt, wobei die Extraktion in einer oder mehreren Stufen erfolgt.Subsequently, the lipophilic components of the virus lysate are extracted. The extractants used are solvents such as phenol, chloroform, isoamyl alcohol or mixtures thereof. Preferably, a mixture of phenol and chloroform / isoamyl alcohol is first used, wherein the extraction takes place in one or more stages.
Weitere Methoden zur Isolierung der Virus-DNA sind z.B. Zentrifugation eines Viruslysats in einem CsCI-Dichtegradienten oder in der Gelelektrophorese (s. Lit. #14).Other methods of isolating the viral DNA are e.g. Centrifugation of a viral lysate in a CsCl density gradient or in gel electrophoresis (see Ref. # 14).
Die Extraktion von Nukleinsäuren wird in Lit. #13 beschrieben.Extraction of nucleic acids is described in Ref. # 13.
Die so extrahierte DNA wird bevorzugt aus der wäßrigen Lösung mit z.B. Alkohol, bevorzugt mit Ethanol oder Isopropanol und unter Zusatz von monovalenten Salzen wie z.B. Alkalichloride oder -acetate, bevorzugt Lithiumchlorid, Natriumchlorid oder Natriumacetat, Kaliumacetat, ausgefällt (s. loc cit.).The DNA so extracted is preferably extracted from the aqueous solution with e.g. Alcohol, preferably with ethanol or isopropanol and with the addition of monovalent salts such. Alkali chlorides or acetates, preferably lithium chloride, sodium chloride or sodium acetate, potassium acetate, precipitated (see loc cit.).
Die so gereinigte virale DNA dient nun der Herstellung von DNA-Fragmenten. Sie wird hierzu beispielsweise mit Restriktionsenzymen behandelt. Geeignete Restriktionsenzyme sind beispielsweise Eco RI, BamH1 HindIII, Kpnl. Alternativ können Genomfragmente mittels Polymerase-Kettenreaktion (PCR) synthetisiert werden. Hierzu werden Primer aus bereits bekannten Sequenzabschnitten des VirusGenoms ausgewählt und mittels beispielsweise Taq bzw. Pfu-Polymerase der vom Primerpaar begrenzte Genomabschnitt in vitro synthetisiert.The purified viral DNA is now used to produce DNA fragments. It is treated for this purpose, for example, with restriction enzymes. Suitable restriction enzymes are, for example, Eco RI, BamHI Hin dIII, KpnI. Alternatively, genome fragments can be synthesized by polymerase chain reaction (PCR). For this purpose, primers are selected from already known sequence segments of the virus genome and synthesized by means of, for example, Taq or Pfu polymerase of the primer pair-limited genome section in vitro.
Die aus Restriktionsverdau bzw. PCR resultierenden DNA-Fragmente können nach den bei (Sambrock 89) beschriebenen Methoden in Vektorsysteme kloniert werden. Beispielsweise bieten sich hierzu, abhängig von der Größe des jeweilig zu klonierenden DNA-Fragmentes, Plasmidvektoren, Lambdaphagen-Vektoren, bzw. Cosmidvektoren an.The DNA fragments resulting from restriction digestion or PCR can be cloned into vector systems according to the methods described in (Sambrock 89) become. For this purpose, for example, depending on the size of the particular DNA fragment to be cloned, plasmid vectors, lambda phage vectors or cosmid vectors are suitable.
In Vektoren klonierte Genomfragmente werden zunächst durch Sequenzierung analysiert. Die inserierten DNA-Fragmente werden mittels verschiedener Restriktionsenzyme kartiert und geeignete Subfragmente in Plasmidvektoren kloniert. Die Sequenzierungsreaktion erfolgt beispielsweise unter Verwendung des T7-Sequencing Kit der Firma Pharmacia nach Angaben des Herstellers. Die hierzu benötigte doppelsträngige Plasmid-DNA wird vorzugsweise nach der PEG-Methode (Hattori und Sakaki 1985) präpariert. Mittels Computeranalyse (CGC, s. vorne) werden auf den Genomfragmenten vorhandene "Offene Leserahmen (ORF)" identifiziert. Erkenntnisse über die jeweilige Funktion der identifizierten ORFs können durch Vergleich ihrer Sequenz mit anderen in einer Datenbank vorhandenen Gensequenzen bekannter Funktion gewonnen werden. Funktionell charakterisiert werden die identifizierten ORFs durch Nachweis ihrer jeweiligen korrespondierenden Transkripte in Virus-infizierten Zellen. Hierzu wird die Gesamt-RNA virus-infizierter Zellen beispielsweise mittels der AGPC-Methode (Chomczynski und Sacchi, (20) 1987) isoliert. Durch Northern Blotanalyse bzw. Primer-Extension und RNA-Schutzversuchen können die spezifischen Transkripte samt ihrer 5'- und 3'-Enden identifiziert werden. Alternativ besteht die Möglichkeit, das durch einen identifizierten ORF kodierte Virusprotein in vitro zu exprimieren, mittels des Expressionsproduktes Antiseren zu gewinnen und die Expression des ORFs nachzuweisen.Genomic fragments cloned into vectors are first analyzed by sequencing. The inserted DNA fragments are mapped by means of various restriction enzymes and suitable subfragments are cloned into plasmid vectors. The sequencing reaction is carried out, for example, using the T7 sequencing kit from Pharmacia according to the manufacturer. The double-stranded plasmid DNA required for this purpose is preferably prepared by the PEG method (Hattori and Sakaki 1985). By means of computer analysis (CGC, see above), "open reading frames (ORF)" present on the genome fragments are identified. Insights into the respective function of the identified ORFs can be obtained by comparing their sequence with other gene sequences of known function present in a database. Functionally, the identified ORFs are characterized by detection of their respective corresponding transcripts in virus-infected cells. For this purpose, the total RNA of virus-infected cells is isolated, for example, by the AGPC method (Chomczynski and Sacchi, (20) 1987). By Northern blot analysis or primer extension and RNA protection experiments, the specific transcripts can be identified together with their 5 'and 3' ends. Alternatively, it is possible to express the virus protein encoded by an identified ORF in vitro, to obtain antisera by means of the expression product and to detect the expression of the ORF.
Um festzustellen, ob das gefundene Gen nicht-essentiell für die Virusvermehrung ist, kann das Gen durch Gendisruption bzw. Gendeletion zerstört werden. Hierbei werden entweder das gesamte Gen bzw. Teile davon aus dem PPV-Genom entfernt oder der Leserahmen des Gens durch die Insertion von Fremdgensequenzen unterbrochen. Das resultierende Virus wird auf Vermehrungsfähigkeit untersucht. Kann das Virus auch ohne die Existenz des zerstörten Genes replizieren, handelt es sich hierbei um ein nicht-essentielles Gen.To determine whether the gene found is non-essential for virus replication, the gene can be destroyed by gene disruption or gene deletion. In this case, either the entire gene or parts thereof are removed from the PPV genome or the reading frame of the gene is interrupted by the insertion of foreign gene sequences. The resulting virus is tested for reproductive ability. Can the virus even without the Replicating the existence of the destroyed gene, this is a non-essential gene.
Welche der oben erhaltenen Klone mit Genomfragmenten des PPV eingesetzt werden, hängt davon ab, ob die herzustellenden rekombinanten PPV (i) replikationsfähig oder (ii) replikationsdefekt sein sollen.
- i. Sollen rekombinante PPV hergestellt werden, die trotz der Insertion und/oder Deletion replikationsfähig sind, werden klonierte virale Genomfragmente weiterverarbeitet, die Gene oder Genombereiche außerhalb von Genen enthalten, die nicht-essentiell für die Virusvermehrung sind oder Genduplikationen enthalten.
Die Prüfung, ob es sich bei dem vorliegenden Gen oder Genombereich um eine nicht-essentielle Region des Virusgenoms oder um eine Genduplikation handelt, wird über Virusmutanten bestimmt. Hierzu wird das untersuchte Gen bzw. der untersuchte Genombereich im PPV durch molekularbiologische Methoden inaktiviert, beispielsweise durch teilweise oder vollständige Deletion des fraglichen Bereiches, und die Vermehrungsfähigkeit der Virusmutante untersucht. Kann die Virusmutante sich trotz der Inaktivierung des fraglichen Gens oder Genombereiches vermehren, handelt es sich bei dem untersuchten Gen oder Genombereich um eine nicht-essentielle Region.
Bevorzugt werden klonierte Genomfragmente des PPV, die die nicht-essentiellen Gene vollständig enthalten. Darüber hinaus sollten an beiden Enden der Gene oder der Genombereiche die flankierenden viralen Genombereiche ebenfalls enthalten sein. Die Länge der flankierenden Bereiche sollte mehr als 100 Basenpaare betragen. Liegen solche Genomklone nicht vor, können sie aus bestehenden Genomklonen mit molekularbiologischen Methoden hergestellt werden. Enhalten die klonierten Genomfragmente zusätzliche Genombereiche, die für die vorliegende Herstellung nicht benötigt werden, können diese durch Subklonierungen entfernt werden. - ii. Sollen rekombinante PPV hergestellt werden, die aufgrund der Insertion und/oder Deletion die Fähigkeit zur Bildung von infektiösen Nachkommen verloren haben, werden klonierte virale Genomfragmente weiterverarbeitet, die Gene oder Genombereiche außerhalb von Genen enthalten, die essentiell für die Virusvermehrung sind.
Die Prüfung, ob es sich bei dem vorliegenden Gen oder Genombereich um eine essentielle Region des Virusgenoms handelt, kann über Virusmutanten bestimmt werden. Hierzu wird das untersuchte Gen bzw. der untersuchte Genombereich im PPV durch molekularbiologische Methoden inaktiviert, beispielsweise durch teilweise oder vollständige Deletion des fraglichen Bereiches, und die Vermehrungsfähigkeit der Virusmutante untersucht. Kann die Virusmutante sich aufgrund der Inaktivierung des fraglichen Gens oder Genombereiches nicht mehr vermehren, handelt es sich bei dem untersuchten Gen oder Genombereich um eine essentielle Region.
Bevorzugt werden klonierte Genomfragmente des PPV, die die essentiellen Gene vollständig enthalten. Darüber hinaus sollten an beiden Enden der Gene oder der Genombereiche die flankierenden viralen Genombereiche ebenfalls enthalten sein. Die Länge der flankierenden Bereiche sollte mehr als 100 Basenpaare betragen. Liegen solche Genomklone nicht vor, können sie aus bestehenden Genomklonen mit molekularbiologischen Methoden hergestellt werden. Enhalten die klonierten Genomfragmente zusätzliche Genombereiche, die für die vorliegende Herstellung nicht benötigt werden, können diese durch Subklonierungen entfernt werden.
- i. If recombinant PPVs are to be produced which are capable of replication despite the insertion and / or deletion, cloned viral genome fragments are further processed which contain genes or genomic regions outside of genes which are non-essential for virus propagation or contain gene duplications.
The test of whether the gene or genome region in question is a non-essential region of the virus genome or a gene duplication is determined by virus mutants. For this purpose, the investigated gene or the investigated genome region in the PPV is inactivated by molecular biological methods, for example by partial or complete deletion of the region in question, and examined the proliferation of the virus mutant. If the virus mutant can multiply despite the inactivation of the gene or genome region in question, the gene or genome region examined is a non-essential region.
Preference is given to cloned genome fragments of the PPV which completely contain the nonessential genes. In addition, flanking viral genomic regions should also be included at both ends of the genes or genomic regions. The length of the flanking areas should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing genome clones using molecular biological methods. If the cloned genome fragments contain additional genome regions which are not suitable for the present preparation are needed, they can be removed by subcloning. - ii. If recombinant PPVs are to be produced which have lost the ability to form infectious offspring due to insertion and / or deletion, cloned viral genome fragments are further processed which contain genes or genomic regions outside of genes which are essential for virus replication.
The test as to whether the gene or genome region in question is an essential region of the viral genome can be determined by virus mutants. For this purpose, the investigated gene or the investigated genome region in the PPV is inactivated by molecular biological methods, for example by partial or complete deletion of the region in question, and examined the proliferation of the virus mutant. If the virus mutant can no longer multiply due to the inactivation of the gene or genome region in question, the gene or genome region examined is an essential region.
Preference is given to cloned genome fragments of the PPV that completely contain the essential genes. In addition, flanking viral genomic regions should also be included at both ends of the genes or genomic regions. The length of the flanking areas should be more than 100 base pairs. If such genome clones are not available, they can be prepared from existing genome clones using molecular biological methods. If the cloned genomic fragments contain additional genome regions which are not required for the present preparation, these can be removed by subcloning.
Soll die ITR-Region, das VEGF-Gen, das PK-Gen, das Gen, das für 10kDa-Protein kodiert oder der intergenische Bereich zwischen dem PK-Gen und dem HD1R-Gen als Insertionsstelle in einem PPV benutzt werden, müssen die entsprechenden Bereiche des PPV-Genoms, die die Insertionsstellen beinhalten, isoliert werden. Hierzu werden die entsprechenden Bereiche des PPV-Genoms kloniert.If the ITR region, the VEGF gene, the PK gene, the gene coding for 10kDa protein or the intergenic region between the PK gene and the HD1R gene is to be used as an insertion site in a PPV, the corresponding Regions of the PPV genome, which include the insertion sites isolated. For this purpose, the corresponding regions of the PPV genome are cloned.
Das Gen, das für VEGF kodiert, wird auf dem Genom des PPV lokalisiert und anschließend in Teilen oder in seiner Gesamtheit mit seinen flankierenden Genom-Abschnitten isoliert. Hierzu wird bevorzugt das PPV gemäß #1 vermehrt und das Genom gemäß #2.1.1 gereinigt.
- a. Das VEGF-Gen wird beispielsweise über eine Polymerase-KettenReaktion (PCR) amplifiziert. Die für diese Reaktion notwendigen Start-Sequenzen (Primer) werden aus der im Sequenz Protokoll ID No: 1 dargestellten DNA-Sequenz des VEGF-Gens abgeleitet. Das erhaltene Amplifikat wird bevorzugt anschließend kloniert.
- b. Bevorzugt wird die Region, die das VEGF-Gen und seine flankierenden Genomabschnitte enthält, über Fragmentierung des PPV-Genoms und Isolierung sowie Klonierung des oder der entsprechenden Genom-Fragmente gewonnen. Hierfür wird das gereinigte Genom des Virus wie in #2.1.2 beschrieben, bevorzugt mit dem Restriktionsenzym HindIII, geschnitten. Zur Identifizierung des oder der Genomfragmente, das oder die das VEGF-Gen und seine flankierenden Genomabschnitte tragen, werden bevorzugt die nach dem Enzymverdau erhaltenen Genom-Fragmente durch elektrophoretische oder chromatographische Verfahren aufgetrennt.
Elektrophoretische Auftrennungen in Agarose bzw. Polyacrylamid werden nach Standardmethoden durchgeführt, die in- Current Protocols in Molecular Biology 1987-1988, Verlag Wiley-Interscience,1987
- A Practical Guide to Molecular Cloning, Perbal, 2nd edition, Verlag Wiley Interscience,1988
- Molecular Cloning, loc. cit.
- Virologische Arbeitsmethoden, Band III, Gustav Fischer Verlag, 1989
Die Identifizierung der Genom-Fragmente, die das VEGF-Gen und seine flankierenden Sequenzen tragen, erfolgt beispielsweise durch Hybridisierung mit definierten Nukleinsäuresonden. Die aufgetrennten Genomfragmente werden hierzu auf Filter übertragen und mit VEGF-spezifischen und markierten Nukleinsäure-Sonden nach der Southern Blotmethode hybridisiert. Die Methode des Transfers der Genomfragmente und der Hybridisierung kann nach Standard-Protokollen, wie sie unter "Southern Blotting" in Molecular Cloning loc.cit. beschrieben sind, erfolgen. Die als Sonden einsetzbaren Oligonukleotide oder Nukleinsäurefragmente lassen sich aus Sequenzprotokoll Seq ID No: 1 ableiten. Beipielsweise wird das TaqI-Subfragment (366 bp), das mittels Seq ID No: 1 identifiziert werden kann als Hybridisierungssonde eingesetzt.
Die Genomfragmente, die nachgewiesenermaßen Teile oder bevorzugt das gesamte VEGF-Gen und die flankierenden Genomabschnitte enthalten, werden isoliert und kloniert. Die elektrophoretische Isolierung des oder der entsprechenden Genomfragmente erfolgt beispielsweise über Elektroelution oder mittels "Low-Melting-Agarose"-Verfahren aus dem entsprechenden Gelbereich.
- a. The VEGF gene is amplified, for example, via a polymerase chain reaction (PCR). The starting sequences (primers) necessary for this reaction are derived from the DNA sequence of the VEGF gene shown in the sequence protocol ID No: 1. The amplicon obtained is preferably subsequently cloned.
- b. Preferably, the region containing the VEGF gene and its flanking genome segments is obtained via fragmentation of the PPV genome and isolation and cloning of the corresponding genome fragment (s). For this, the purified genome of the virus is cut as described in # 2.1.2, preferably with the restriction enzyme HindIII. To identify the genome fragment (s) carrying the VEGF gene and its flanking genome segments, preferably the genome fragments obtained after enzyme digestion are separated by electrophoretic or chromatographic techniques.
Electrophoretic separations in agarose or polyacrylamide are carried out according to standard methods which are described in- Current Protocols in Molecular Biology 1987-1988, published by Wiley-Interscience, 1987
- A Practical Guide to Molecular Cloning, Perbal, 2 nd edition, Wiley Interscience, 1988
- Molecular Cloning, loc. cit.
- Virological Working Methods, Volume III, Gustav Fischer Verlag, 1989
The identification of the genome fragments carrying the VEGF gene and its flanking sequences, for example, by hybridization with defined nucleic acid probes. For this purpose, the separated genomic fragments are transferred to filters and hybridized with VEGF-specific and labeled nucleic acid probes according to the Southern blot method. The method of transfer of genomic fragments and hybridization can be carried out according to standard protocols as described under "Southern blotting" in Molecular Cloning loc. are described done. The oligonucleotides or nucleic acid fragments which can be used as probes can be derived from Sequence Listing Seq ID No: 1. For example, the TaqI subfragment (366 bp), which can be identified by Seq ID No: 1, is used as the hybridization probe.
The genomic fragments that have been shown to contain portions or preferably the entire VEGF gene and the flanking genome segments are isolated and cloned. The electrophoretic isolation of the corresponding genome fragments or, for example, by electroelution or by "low-melting agarose" method from the corresponding gel area.
Zur Klonierung des VEGF-Gens werden die oben hergestellten Genom-Fragmente in bakterielle oder eukaryontische Vektoren inseriert. Besonders bevorzugt werden zunächst bakterielle Plasmid- oder Phagen-Vektoren. Zur Insertion des Genomfragmentes werden doppelsträngige Plasmid- oder Phagenvektor-DNA-Moleküle mit Restriktionsenzymen behandelt, so daß für die Insertion geeignete Enden entstehen.For cloning the VEGF gene, the genomic fragments prepared above are inserted into bacterial or eukaryotic vectors. Particularly preferred are first bacterial plasmid or phage vectors. For insertion of the genomic fragment, double-stranded plasmid or phage vector DNA molecules are treated with restriction enzymes to form suitable ends for insertion.
Als Plasmide dienen bekannte Plasmide, wie z.B. pBR322 und seine Abkömmlinge, z.B. pSPT18/19, pAT153, pACYC184, pUC18/19, pSP64/65.Plasmids are known plasmids, e.g. pBR322 and its derivatives, e.g. pSPT18 / 19, pAT153, pACYC184, pUC18 / 19, pSP64 / 65.
Als Phagenvektoren dienen z.B. die bekannten Varianten des Phagen Lambda wie z.B. -ZAP, -gt 10/11 oder Phage M13mp18/19.As phage vectors are used e.g. the known variants of the phage lambda, e.g. -ZAP, -gt 10/11 or phage M13mp18 / 19.
Die einsetzbaren Restriktionsenzyme sind bekannt z.B. aus Gene Band 92 (1989) Elsevier Science Publishers BV Amsterdam.The usable restriction enzymes are known e.g. from Gene Vol. 92 (1989) Elsevier Science Publishers BV Amsterdam.
Das mit Restriktionsenzym behandelte Plasmid oder der Phagenvektor wird mit einem Überschuß des zu inserierenden DNA-Fragments, z.B. etwa im Verhältnis 5:1, gemischt und mittels DNA-Ligasen behandelt, in den Vektor ligiert. Der Ligationsansatz wird zur Vermehrung der Plasmide oder Phagen in pro- oder eukaryontischen Zellen, bevorzugt in Bakterien (z.B. Escherichia coli Stamm K-12 und seine Derivate) eingebracht und diese vermehrt.The restriction enzyme treated plasmid or phage vector is incubated with an excess of the DNA fragment to be inserted, e.g. in the ratio 5: 1, mixed and treated by means of DNA ligases, ligated into the vector. The ligation mixture is introduced into and propagates the plasmids or phages in prokaryotic or eukaryotic cells, preferably in bacteria (e.g., Escherichia coli strain K-12 and its derivatives).
Transformation und Selektion der Bakterien erfolgt wie in Molecular Cloning loc. sit. beschrieben.Transformation and selection of the bacteria is carried out as described in Molecular Cloning loc. sit. described.
Die Identität der Fremd-DNA wird bevorzugt über Hybridierungsexperimente und besonders bevorzugt über Sequenzanalysen verifiziert. Gegebenenfalls werden Subklonierungen vorgenommen.The identity of the foreign DNA is preferably verified via hybridization experiments and more preferably via sequence analyzes. If necessary, subcloning is carried out.
Das Gen, das für die Proteinkinase kodiert, wird auf dem Genom des PPV lokalisiert und anschließend in Teilen oder ganz mit seinen flankierenden Genomabschnitten isoliert.The gene encoding the protein kinase is localized on the genome of the PPV and subsequently isolated in part or all with its flanking genome segments.
Dieser Bereich kann, wie für die Klonierung des VEGF-Gens beschrieben. über die Fragmentierung des PPV-Genoms (Spaltstellen siehe Abb. 1). bevorzugt mit anschließender Klonierung der Fragmente, und Selektion der Fragmente bzw. Klone, die Teile des PK-Gens oder bevorzugt das gesamte PK-Gen mit flankierenden DNA-Sequenzen des PPV-Genoms enthalten, gewonnen werden. DNA-Moleküle, die als Primer einer PCR oder als Sonden für eine Hybridisierung eingesetzt werden können, lassen sich aus der Sequenz aus Protokoll ID No: 2 bzw. ID No: 9 ableiten.This area can be described as for the cloning of the VEGF gene. on the fragmentation of the PPV genome (cleavage sites see Fig. 1). preferably with subsequent cloning of the fragments, and selection of the fragments or clones which contain parts of the PK gene or preferably the entire PK gene with flanking DNA sequences of the PPV genome are obtained. DNA molecules used as primers of a PCR or as Probes can be used for a hybridization, can be derived from the sequence of Protocol ID No: 2 or ID No: 9.
Gegebenenfalls werden Subklonierungen vorgenommen.If necessary, subcloning is carried out.
Das Gen, das für das 10kDa-Protein kodiert wird entsprechend dem Verfahren, wie es für das VEGF-Gen und das PK-Gen ausführlich beschrieben wurde (oben), auf dem Genom des PPV lokalisiert und in Teilen oder bevorzugt ganz mit seinen flankierenden PPV-Genomsequenzen isoliert.The gene encoding the 10kDa protein is located on the genome of the PPV and partially or preferably entirely with its flanking PPV, according to the procedure detailed for the VEGF gene and the PK gene (above) Genome sequences isolated.
Dabei wird der Bereich des PPV-Genoms, der das Gen für das 10kDa-Protein oder Teile davon enthält, bevorzugt über PCR und/oder Klonierung und Identifizierung und Selektion der geeigneten Klone erhalten.In this case, the region of the PPV genome containing the gene for the 10kDa protein or parts thereof is preferably obtained by PCR and / or cloning and identification and selection of the appropriate clones.
DNA-Moleküle, die als Primer einer PCR oder als Sonden für eine Hybridisierung eingesetzt werden können, können Lit. #8 entnommen werden. Gegebenenfalls werden Subklonierungen vorgenommen.DNA molecules that can be used as primers of a PCR or as probes for hybridization can be found in reference # 8. If necessary, subcloning is carried out.
Die Vorgehensweise entspricht der ausführlich beschriebenen Klonierung des VEGF-Gens (oben). Die DNA-Moleküle, die zur Isolierung der entsprechenden Bereiche als Primer einer PCR oder als Sonden für eine Hybridisierung eingesetzt werden können, sind im Sequenz Protokoll ID No: 4 (ITR-Region) und No: 7 (Bereich zwischen PK-Gen und HDIR-Gen) ersichtlich.The procedure corresponds to the detailed cloning of the VEGF gene (above). The DNA molecules that can be used to isolate the corresponding regions as primers of a PCR or as probes for hybridization are described in the Sequence Protocol ID No: 4 (ITR region) and No: 7 (region between PK gene and HDIR Gene).
Auf Basis der gemäß Abschnitt 2 identifzierten, lokalisierten und klonierten Genomfragmente von PPV werden sogenannte Insertionsplasmide hergestellt, die zur Insertion von Fremd-DNA in das Genom der PPV benutzt werden können. Die Insertionsplasmide tragen die in das PPV zu inserierende Fremd-DNA, flankiert von Genomabschnitten des PPV. Es gibt verschiedene Möglichkeiten, die Insertionsplasmide herzustellen: Als Beispiele seien hier erwähnt:Based on the localized and cloned genomic fragments of PPV identified in accordance with
Nur einmal vorkommende, d.h. singuläre Restriktionsschnittstellen, lassen sich beispielsweise (s. 2.1.3) den ermittelten PPVNukleotidsequenzen identifizieren.Only one-time, i. singular restriction sites, for example (see 2.1.3) can be identified the identified PPV-nucleotide sequences.
In diese können synthetisch hergestellte Oligonukleotide, die neue singuläre Schnittstellen für Restriktionsenzyme tragen, eingebaut werden.Synthesized oligonucleotides carrying new unique restriction enzyme cleavage sites can be incorporated into these.
Die erhaltenen Plasmide werden wie zuvor beschrieben vermehrt und selektiert.The resulting plasmids are propagated and selected as described above.
Alternativ können mittels "PCR", wie von Jacobs et al. (Lit. #12) beschrieben, neue singuläre Restriktionsenzymerkennungsstellen in die PPV-Genomfragmente eingebaut werden .Alternatively, by "PCR" as described by Jacobs et al. (Ref. # 12), new unique restriction enzyme recognition sites are incorporated into the PPV genome fragments.
Die identifizierten und/oder hergestellten singulären Restriktionsenzymerkennungsstellen dienen zur Insertion von Fremd-DNA in das PPV-Genom.The identified and / or produced unique restriction enzyme recognition sites serve to insert foreign DNA into the PPV genome.
Die Insertion der Fremd-DNA erfolgt durch bekannte Methoden (Lit. #11).The insertion of the foreign DNA is carried out by known methods (Ref. # 11).
Die Deletion von Subfragmenten aus den klonierten PPV-Genomfragmenten kann beispielsweise durch Behandlung mit Restriktionsenzymen erfolgen, die bevorzugt mehr als eine, besonders bevorzugt 2 Erkennungsstellen besitzen. Nach der Enzymbehandlung werden die erhaltenen Fragmente, wie oben beschrieben beispielsweise elektrophoretisch aufgetrennt, isoliert und die entsprechenden Fragmente durch Ligase-Behandlung wieder zusammengefügt. Die erhaltenen Plasmide werden vermehrt und die deletierten Plasmide selektiert.The deletion of subfragments from the cloned PPV genome fragments can be carried out, for example, by treatment with restriction enzymes which preferably have more than one, particularly preferably 2, recognition sites have. After the enzyme treatment, the fragments obtained are isolated, as described above, for example by electrophoresis, and the corresponding fragments are reassembled by ligase treatment. The resulting plasmids are propagated and the deleted plasmids are selected.
Alternativ wird eine singuläre Restriktionsenzymerkennungsstelle auf dem PPV-Genomfragment als Startpunkt für einen bidirektionalen Abbau der Fragmente mit einer Endonuklease, beispielsweise dem Enzym Bal31, genutzt. Die Größe der Deletion kann durch die Dauer der Einwirkung des Enzyms bestimmt werden und gelelektrophoretisch überprüft werden. An die neu entstandenen Fragmentenden werden synthetische Oligonukleotide, wie unter 3.1 (oben) beschrieben, ligiert werden.Alternatively, a unique restriction enzyme recognition site on the PPV genomic fragment is used as a starting point for bidirectional degradation of the fragments with an endonuclease, for example the enzyme Bal31. The size of the deletion can be determined by the duration of the action of the enzyme and checked by gel electrophoresis. Synthetic oligonucleotides, as described under 3.1 (above), are ligated to the newly formed fragment ends.
Die Fremdgenübertragung in das PPV-Genom gelingt nur bei einem geringen Prozentsatz der Gesamt PPV-Population.Foreign gene transfer into the PPV genome is only successful in a small percentage of the total PPV population.
Deshalb sind zur Trennung rekombinanter vom Wildtyp-PPV Selektionssysteme nötig (Lit. #Moss).Therefore, for the separation of recombinant from wild-type PPV selection systems are necessary (Ref # Moss).
Bevorzugt wird das gpt-Selektionssystem eingesetzt, welches auf dem Guanyl-Phosphoribosyl-Transferasegen von E.coli basiert. Dieses Gen vermittelt, exprimiert in einer eukaryotischen Zelle, Resistenz gegen Mycophenolsäure, einen Inhibitor des Purin-Metabolismus. Sein Einsatz bei der Konstruktion rekombinanter Vektor-Viren ist mehrfach beschrieben (s. Lit. #16 / #17).Preferably, the gpt selection system is used, which is based on the guanyl phosphoribosyl transferase gene of E. coli. This gene, expressed in a eukaryotic cell, mediates resistance to mycophenolic acid, an inhibitor of purine metabolism. Its use in the construction of recombinant vector viruses has been described several times (see reference # 16 / # 17).
Die Insertion von Fremd-DNA in das PPV-Genom erfolgt durch:
- a. Gleichzeitige Transfektion der Insertions- oder Deletionsplasmid-DNA und Infektion mit PPV in geeigneten Wirtszellen,
- b. Transfektion der Insertions- oder Deletionsplasmid- DNA und anschließende Infektion mit dem PPV in geeigneten Wirtszellen,
- c. Infektion mit dem PPV und anschließende Transfektion mit der Insertions-oder Deletionsplasmid-DNA in geeigneten Wirtszellen.
- a. Simultaneous transfection of the insertion or deletion plasmid DNA and infection with PPV in appropriate host cells,
- b. Transfection of the insertion or deletion plasmid DNA and subsequent infection with the PPV in suitable host cells,
- c. Infection with the PPV and subsequent transfection with the insertion or deletion plasmid DNA in appropriate host cells.
Die Methoden der hierzu geeigneten Verfahren sind bekannt. Die Transfektion kann mittels bekannter Methoden erfolgen wie z.B. Calcium-Phosphat-Technik, Liposomen-vermittelte Transfektion oder Elektroporation (s. Lit. #18).
- 1. Infektion mit PPV:
- Für die Herstellung der PPV mit Fremd-DNA werden Zellkulturen bevorzugt, die eine gute Virusvermehrung und eine effiziente Transfektion erlauben, beispielsweise die permanente Rindernierenzelle BK-K1-3A.
- 2. Herstellung der Insertions- oder Deletionsplasmid-DNA:
- Die nach den vorher beschriebenen Verfahren erhaltenen transformierten Zellen, beispielsweise Bakterien, die Insertions-oder Deletionsplasmide enthalten, werden vermehrt und die Plasmide in an sich bekannter Weise aus den Zellen isoliert und weiter gereinigt. Die Reinigung erfolgt z.B. durch eine isopyknische Zentrifugation im Dichtegradienten von z.B. CsCl. oder durch Affinitätsreinigung an kommerziell erhältlichen Silikapartikeln.
- 3. Transfektion:
- Zur Transfektion wird bevorzugt gereinigte Plasmid-DNA zirkular oder linearisiert eingesetzt. Die Reinigung erfolgt z.B. wie unter Abschnitt 2 (oben) angegeben.
- 4. Kultivierung transfizierter und infizierter Zellen
Die Zellen werden nach den oben beschriebenen Methoden kultiviert. Beim Auftreten eines zytopathogenen Effektes wird das Kulturmedium entfernt, gegebenenfalls durch Zentrifugation oder Filtration von Zelltrümmern befreit und gegebenenfalls gelagert sowie nach den herkömmlichen Methoden der Einzel-Plaque-Reinigung von Viren aufgearbeitet.
- 1. Infection with PPV:
- For the production of PPV with foreign DNA, cell cultures are preferred which allow good virus replication and efficient transfection, for example the permanent bovine kidney cell BK-K1-3A.
- 2. Preparation of Insertion or Deletion Plasmid DNA:
- The transformed cells obtained by the previously described methods, for example bacteria which contain insertion or deletion plasmids, are propagated and the plasmids are isolated from the cells in a manner known per se and further purified. The purification is carried out, for example, by an isopycnic centrifugation in the density gradient of, for example, CsCl. or by affinity purification on commercially available silica particles.
- 3. Transfection:
- For transfection, purified plasmid DNA is preferably used in a circular or linearized manner. The cleaning takes place eg as described in section 2 (above).
- 4. Cultivation of transfected and infected cells
The cells are cultured according to the methods described above. When a cytopathogenic effect occurs, the culture medium is removed, optionally freed by centrifugation or filtration of cell debris and optionally stored and worked up by the conventional methods of single-plaque purification of viruses.
Folgendes Verfahren wird bei der Herstellung rekombinanter PPV angewandt:The following procedure is used in the production of recombinant PPV:
Konfluent ausgewachsene BK-KL-3A Zellen werden mit einer Infektionsdosis von 0,001 bis 5, bevorzugt 0,1 MOI (multiplicity of infection) infiziert. Zwei Stunden später werden die infizierten Zellen beispielsweise mit DNA (2-10 µg) des Plasmids pMT-10 entweder nach der CaPO4-Gly-cerolschock-Methode oder unter Verwendung eines Transfektions-Kits nach Angaben des Herstellers (DOSPER, Boehringer-Manaheim) transfiziert. Anschließend werden diese Zellkulturen mit Medium während drei bis sechs Tagen bei 37°C, 5 % CO2-Atmosphäre inkubiert, bis cpe oder Plaquebildung sichtbar wurde.Confluent adult BK-KL-3A cells are infected with an infection dose of 0.001 to 5, preferably 0.1 MOI (multiplicity of infection). Two hours later, the infected cells are, for example, with DNA (2-10 ug) of the plasmid pMT-10 either by the CaPO 4 glycerol shock method or using a transfection kit according to the manufacturer (DOSPER, Boehringer-Manaheim) transfected. Subsequently, these cell cultures are incubated with medium for 3 to 6 days at 37 ° C, 5% CO 2 atmosphere until cpe or plaque formation is visible.
Die Identifizierung von rekombinanten PPV erfolgt in Abhängigkeit von der inserierten Fremd-DNA z.B. durch:
- a. Nachweis der Fremd-DNA, z.B. durch DNA/DNA-Hybridisierungen
- b. Die Amplifikation der Fremd-DNA mittels der PCR
- c. Expression der Fremd-DNA mit Hilfe der rekombinanten Viren
- a. Detection of foreign DNA, eg by DNA / DNA hybridizations
- b. The amplification of the foreign DNA by PCR
- c. Expression of foreign DNA using the recombinant viruses
Hierzu wird die DNA aus dem fraglichen Virus gewonnen und mit Nukleinsäure, die zumindest in Teilen mit der inserierten Fremd-DNA identisch ist, hybridisiert.For this purpose, the DNA is obtained from the virus in question and hybridized with nucleic acid which is identical at least in part to the inserted foreign DNA.
Die Einzel-Plaque-gereinigten und als Rekombinanten identifizierten PPV werden bevorzugt nochmals auf Vorhandensein und/oder Expression der Fremd-DNA geprüft. Rekombinante PPV, die die Fremd-DNA stabil enthalten und/oder exprimieren, stehen für die weitere Anwendung zur Verfügung.The individual plaque-purified and identified as recombinants PPV are preferably tested again for the presence and / or expression of the foreign DNA. Recombinant PPV stably containing and / or expressing the foreign DNA are available for further use.
Die Expression der Fremd-DNA auf Protein-Ebene kann nachgewiesen werden durch z.B. Infektion von Zellen mit dem Virus und anschließender Immunfluoreszenzanalyse mit spezifischen Antikörpern gegen das von der Fremd-DNA kodierte Protein oder durch Immunpräzipitation oder Western Blotting mit Antikörpern gegen das von der Fremd-DNA kodierte Protein aus den Lysaten infizierter Zellen.The expression of the foreign DNA at the protein level can be detected by, for example, infection of cells with the virus and subsequent immunofluorescence analysis with specific antibodies to that of the Foreign DNA encodes protein or by immunoprecipitation or Western blotting with antibodies against the foreign DNA encoded protein from the lysates of infected cells.
Die Expression der Fremd-DNA auf RNA-Ebene kann durch Nachweis der spezifischen Transkripte erfolgen. Hierzu wird RNA aus Virus infizierten Zellen isoliert und mit einer DNA-Sonde hybridisiert, die zumindest in Teilen identisch ist mit der inserierten Fremd-DNA.Expression of foreign DNA at the RNA level can be accomplished by detection of the specific transcripts. For this purpose, RNA is isolated from virus-infected cells and hybridized with a DNA probe which is at least partially identical to the inserted foreign DNA.
Die folgenden Beispiele beschreiben einen Bereich des PPV-Genoms, der sich für die Insertion und Expression homologer und heterologer Gene oder Teilen davon eignet. Geeignete genomische Fragmente sind auf dem HindIII-Fragment I des PPV ovis-Stammes D1701 (und seiner jeweiligen Derivate) enthalten (Sequenz Protokoll ID No: 8 und ID No: 12).The following examples describe a region of the PPV genome suitable for the insertion and expression of homologous and heterologous genes or parts thereof. Suitable genomic fragments are contained on HindIII fragment I of PPV ovis strain D1701 (and its respective derivatives) (Sequence Protocol ID No: 8 and ID No: 12).
Nach Spaltung gereinigter viraler DNA mit dem Restriktionsenzym HindIII wurden die resultierenden DNA-Fragmente durch Agarosegel-Elektrophorese getrennt, das etwa 5,6 kbp große Fragment I ausgeschnitten und mittels der Qiaex®-Methode (Qiagen) isoliert und gereinigt. Dieses DNA-Fragment wurde mit Standardtechniken (Maniatis et al.) in das mit HindIII gespaltene und mit CIP (Kälberdarm-Phosphatase) behandelte Vektorplasmid pSPT18 (Boehringer, Mannheim) kloniert. Die resultierenden rekombinanten Plasmide pORF-1 und pORF-2 unterscheiden sich nur durch die Orientierung des Inserts. Die Anfertigung einer Restriktionskarte ermöglichte eine weitere Subklonierung, wie es in Abb. 1 gezeigt wird. Alle rekombinanten Plasmid-DNAs wurden durch Southern-Blot-Hybridisierung auf restriktionsverdaute virale oder Plasmid-DNA getestet, um ihre Identität und virale Herkunft zu überprüfen.The resulting DNA fragments were separated by agarose gel electrophoresis after cleavage of purified viral DNA with the Hind III restriction enzyme, the excised fragment of about 5.6 kbp and I by means of the Qiaex ® method (Qiagen) and purified. This DNA fragment was cloned by standard techniques (Maniatis et al.) Into HindIII digested and CIP (calf intestinal phosphatase) treated vector plasmid pSPT18 (Boehringer, Mannheim). The resulting recombinant plasmids pORF-1 and pORF-2 differ only in the orientation of the insert. The preparation of a restriction map allowed further subcloning, as shown in FIG. All recombinant plasmid DNAs were tested by Southern blot hybridization for restriction digested viral or plasmid DNA to verify their identity and viral origin.
Die DNA-Sequenzierung wurde durch Verwendung von doppelsträngiger DNA der verschiedenen rekombinanten Plasmide und SP6- sowie T7-spezifischen Primern, die an beide Enden der Klonierungsstelle des Vektorplasmids pSPT18 binden, bewerkstelligt. Das Didesoxy-Kettenterminationsverfahren nach Sanger wurde nach den Empfehlungen des Herstellers (Pharmacia - Biotech) in Gegenwart von 35S-[α]-dATP und T7-DNA-Polymerase durchgeführt. Gemäß der erhaltenen DNA-Sequenz wurde eine Vielzahl von Oligonukleotiden synthetisiert und zum Sequenzieren beider Stränge des HindIII-Fragments I verwendet. Um Sequenzierungsartefakte oder Bandenkompression aufgrund des relativ hohen G+C-Gehalts des viralen DNA-Inserts (64,78 %) aufzulösen, wurde 7-Desaza-GTP verwendet, und die Sequenzierungsprodukte wurden, falls notwendig, in formamidhaltigen denaturierenden Polyacrylamidgelen getrennt.DNA sequencing was accomplished by using double stranded DNA of the various recombinant plasmids and SP6 and T7 specific primers that bind to both ends of the cloning site of the vector plasmid pSPT18. The Sanger dideoxy chain termination method was performed according to the manufacturer's recommendations (Pharmacia Biotech) in the presence of 35 S- [α] -dATP and T7 DNA polymerase. According to the obtained DNA sequence, a plurality of oligonucleotides were synthesized and used for sequencing both strands of HindIII fragment I. To resolve sequencing artifacts or band compression due to the relatively high content of the viral DNA insert (64.78%), 7-deaza-GTP was used and the sequencing products separated, if necessary, in formamide containing denaturing polyacrylamide gels.
Eine computergestützte Analyse der erhaltenen DNA-Sequenz (Sequenzprotokoll ID No: 8 und ID No: 12) offenbarte mehrere mögliche offene Leserahmen (ORF). Die von diesen ORFs abgeleitete Aminosäuresequenz wurde für Genhomologierecherchen (z.B. Programm GCG) verwendet. Als Ergebnis wurden signifikante Aminosäurehomologien mit den folgenden Genen nachgewiesen (siehe auch Abb. 1).
- 3.1 Ein ORF mit Aminosäurehomologie (36,1
bis 38,3 % Identität; 52,8bis 58,6 % Ähnlichkeit) zum vaskulären endothelialen Wachstumsfaktor (VEGF) verschiedener Säugerspezies (z.B. Maus, Ratte, Meerschweinchen, Rind, Mensch) als auch zu dem VEGF-Gen-Homolog, der vor kurzem in den PPV-Stämmen NZ-2 und NZ-7 beschrieben wurde (Lit. #6), wurde gefunden. Ein entsprechendes Genhomolog ist bei anderen Pockenviren, wie verschiedenen Orthopoxviren, nicht bekannt. Dieser ORF, der als VEGF bezeichnet wird, umfaßt 399 Nucleotide und kodiert ein Polypeptid mit 132 Aminosäuren mit einem berechneten Molekulargewicht von 14,77 kDa. Transkriptionsanalysen unter Verwendung von Northern-Blothybridisierung, RNA-Schutzversuchen und Primerextensionstests mit gesamter oder Oligo (dT) - selektierter RNA, die aus infizierten Zellen isoliert wurde, belegten die Expression von VEGF als frühesGen ab etwa 2 Stunden nach der Infektion (p.i.). Es wurde gefunden, daß die spezifische mRNA 422 bis 425 Basen abdeckt, beginnend direkt stromabwärts einer Sequenz, die 100 % Homologie mit dem kritischen Bereich eines Consensusmotivs zeigt, das typisch für Promotoren früher Gene des Vaccinia-Virus ist. Das 3'-Ende der VEGF-mRNA konnte in eine Consensussequenz kartiert werden, die frühen Transkripten von z.B. Vaccinia-Virus-Genen gemeinsam ist. Durch Northern-Blothybridisierung wurde die Größe dieser mRNA auf ungefähr 500 Basen geschätzt, was auf einen Poly(A)-Abschnitt mit einer Länge von etwa 100 Basen hinweist. - 3.2 Ein weiterer ORF wurde gefunden, der für eine potentielle Proteinkinase (PK) mit Homologie zu einem entsprechenden Gen kodiert, das in mehreren Orthopoxviren (z.B. Vaccinia-, Variola- oder Shope-Fibroma-Virus) vorkommt und als F10L bekannt ist. Dieses Gen-Homolog wird im Infektionszyklus von PPV-Stamm D1701 spät transkribiert (ab 12 bis 16 Stunden p.i.). Der Transkriptionsstartpunkt befindet sich kurz stromabwärts eines Bereichs, der eine hohe Homologie zu bekannten Promotoren später Gene des Vaccinia-Virus zeigt.
- 3.3 Weitere mögliche ORFs wurden gefunden, die bisher keine auffälligen Homologien zu bekannten Gensequenzen zeigen. Von besonderem Interesse ist ein potentielles Gen, das als Gen HD1R bezeichnet wird (Abb. 1). Analysen wie die oben beschriebenen zeigten die Transkription einer spezifischen frühen mRNA mit einer Größe von ca. 1,6 kb.
- 3.4 Schließlich wurde mittels Computer ein ORF gefunden, der das 3'-Ende von F10L und das 5'-Ende von VEGF überlappt (F9L, Abb. 1). Der Sequenzvergleich zeigte Homologie mit dem F9L-Gen des Vaccinia-Virus.
- 3.5 Verglichen zu bekannten DNA Sequenzen der Orf Stämme NZ-2 und NZ-7 ließ sich der Beginn der sogenannten ITR. Region im Genom von D 1701 bei Nukleotidposition 1611 des HindIII-Fragments I festlegen. Bei der ITR-Region handelt es sich um einen am Ende des Pockenvirusgenoms auftretenden Sequenzbereich, der am anderen Genomende in umgekehrter Orientierung ebenfalls vorliegt und daher als "inverted repeat region" (ITR) bezeichnet wird (siehe Sequenzprotokoll ID No: 4). Der Befund des Sequenzvergleichs deckt sich mit Versuchen zur Genomlokalisation des in pORF-1 klonierten Hindin-Fragments I von D1701. Die Kartierung dieses Fragments und des vermutlich identischen HindIII-Fragments H ist in Abb. 2 dargestellt. Hieraus läßt sich schließen, daß die ITR des D1701 Genoms ca. 2,6 kbp umfaßt.
Versuche zur Bestimmung des 3' Endes der VEGF mRNA von D1701 enthüllten, daß zwischen ca. 40 und 220 bp nach dem Übergang zur ITR in der ITR mindestens eine weitere virusspezifische RNA startet. Das entsprechende Gen wurde aufgrund der Aminosäurehomologie zu NZ-2 als ORF3 bezeichnet (Abb. 1). Vor dem putativen 5' Ende der ORF3-mRNA befindet sich eine Konsensussequenz, die typisch für einen frühen Promotor von Pockenviren ist. Homologie zu anderen bekannten Genen konnte bisher nicht gefunden werden.
- 3.1 An ORF with amino acid homology (36.1 to 38.3% identity, 52.8 to 58.6% similarity) to the vascular endothelial growth factor (VEGF) of various mammalian species (eg mouse, rat, guinea pig, bovine, human) as well the VEGF gene homologue recently described in PPV strains NZ-2 and NZ-7 (ref. # 6) was found. A corresponding gene homologue is unknown in other smallpox viruses, such as various orthopoxviruses. This ORF, designated VEGF, comprises 399 nucleotides and encodes a 132 amino acid polypeptide with a calculated molecular weight of 14.77 kDa. Transcriptional analyzes using Northern blot hybridization, RNA protection experiments and primer extension testing with whole or oligo (dT) -selected RNA isolated from infected cells demonstrated expression of VEGF as an early gene from about 2 hours after infection (pi). The specific mRNA was found to cover 422 to 425 bases, starting just downstream of a sequence showing 100% homology with the critical region of a consensus motif typical of promoters of early vaccinia virus genes. The 3 'end of the VEGF mRNA could be mapped into a consensus sequence common to early transcripts of vaccinia virus genes, for example. Northern blot hybridization estimated the size of this mRNA to be approximately 500 bases, indicating a poly (A) segment about 100 bases in length.
- 3.2 Another ORF was found encoding a potential protein kinase (PK) with homology to a corresponding gene found in several orthopoxviruses (eg vaccinia, variola or shope fibroma virus), known as F10L. This gene homologue is late transcribed in the infection cycle of PPV strain D1701 (from 12 to 16 Hours pi). The transcription start site is located just downstream of a region that shows high homology to known promoters of late vaccinia virus genes.
- 3.3 Further possible ORFs have been found which so far show no conspicuous homologies to known gene sequences. Of particular interest is a potential gene called the HD1R gene (Figure 1). Analyzes such as those described above demonstrated the transcription of a specific early mRNA of about 1.6 kb in size.
- 3.4 Finally, an ORF was found by computer overlapping the 3 'end of F10L and the 5' end of VEGF (F9L, Fig. 1). Sequence comparison showed homology with the vaccinia virus F9L gene.
- 3.5 Compared to known DNA sequences of the Orf strains NZ-2 and NZ-7, the so-called ITR began. Region in the genome of D 1701 at nucleotide position 1611 of HindIII fragment I. The ITR region is a sequence region occurring at the end of the poxvirus genome which is also present in reverse orientation at the other end of the genome and is therefore designated as "inverted repeat region" (ITR) (see Sequence Listing ID No: 4). The finding of the sequence comparison coincides with experiments on the genome localization of the pORF-1-cloned Hindin fragment I of D1701. The mapping of this fragment and the probably identical HindIII fragment H is shown in FIG. It can be concluded that the ITR of the D1701 genome comprises about 2.6 kbp.
Experiments to determine the 3 'end of D1701 VEGF mRNA revealed that at least one additional virus-specific RNA starts in the ITR between about 40 and 220 bp after the ITR transition. The corresponding gene was designated ORF3 because of the amino acid homology to NZ-2 (Figure 1). Prior to the putative 5 'end of ORF3 mRNA, there is a consensus sequence that is typical of an early promoter of poxviruses. Homology to other known genes could not be found so far.
Die Möglichkeit der Verwendung des beschriebenen HindIII-DNA-Fragments (in den Plasmiden pORF-1 und pORF-2 kloniert) für die Einführung homologer oder heterologer DNA-Sequenzen wurde erarbeitet. Im folgenden wurden drei verschiedene Strategien angewandt, um dieses Ziel zu erreichen.The possibility of using the HindIII DNA fragment described (cloned in the plasmids pORF-1 and pORF-2) for the introduction of homologous or heterologous DNA sequences was developed. In the following, three different strategies were used to achieve this goal.
Für die folgenden Beispiele wurde das Plasmid pGSRZ konstruiert, das das funktionelle LacZ-Gen von E. coli unter der Kontrolle des 11K-Promotors des Vaccinia Virus enthält. Zu diesem Zweck wurden die relevanten DNA-Teile des Plasmids pUCIILZ (s. Lit. #7) isoliert und in das Plasmid pSPT18 einkloniert. Diese funktionelle 11K-LacZ-Genkombination (im Folgenden als LacZ-Kassette bezeichnet) kann durch Isolierung eines 3,2-kbp großen SmaI-SalI-Fragments von pGSRZ erhalten werden (Abb. 3).For the following examples, the plasmid pGSRZ was constructed which contains the functional LacZ gene of E. coli under the control of the
Ausgehend von den Plasmiden pGSRZ (enthält das LacZ Gen unter der Kontrolle des Vaccinia Viruspromotors P11K), pMT-1 (enthält den PPV VEGF-Promotor) und pMT5 (enthält das E. coli gpt-Gen) wurden verschiedene sogenannte Selektionskassetten konstruiert, wie in Abbildung No: 7 dargestellt. Synthetische komplementäre Oligonukleotide, die die Sequenz des VEGF-Promotors (PVEGF) repräsentieren, wurden hergestellt und in die Smal-Schnittstelle von pSPT18 inseriert (pMT-1). Anschließend wurden die Plasmide pMT-2 und pMT-4 hergestellt, indem das LacZ-Gen aus p18Z (erhalten durch Insertion des BamHI-Fragments aus pGSRZ in pSPT18) bzw. das gpt-Gen aus pMT-5 durch BamHI-Spaltung entfernt und in pMT-1 eingefügt wurde (Abb. 7).Starting from the plasmids pGSRZ (containing the lacZ gene under the control of the vaccinia virus promoter P 11K ), pMT-1 (containing the PPV VEGF promoter) and pMT5 (containing the E. coli gpt gene), various so-called selection cassettes were constructed, such as shown in Figure No: 7. Synthetic complementary oligonucleotides representing the sequence of the VEGF promoter (P VEGF ) were prepared and inserted into the SmaI site of pSPT18 (pMT-1). Subsequently, plasmids pMT-2 and pMT-4 were prepared by removing the LacZ gene from p18Z (obtained by insertion of the BamHI fragment from pGSRZ into pSPT18) or the gpt gene from pMT-5 by BamHI digestion, and into pMT-1 was inserted (Figure 7).
Das funktionelle gpt-Gen wurde mittels PCR von dem gpt-Plasmid pMSG (Pharmacia-Biotech) amplifiziert und in den Vektor pCRII durch sogenannte TA-Klonierung entsprechend den Angaben des Herstellers kloniert (Invitrogen Inc.).The functional gpt gene was amplified by PCR from the gpt plasmid pMSG (Pharmacia Biotech) and cloned into the vector pCRII by so-called TA cloning according to the instructions of the manufacturer (Invitrogen Inc.).
Im folgenden konnten, wie in Abb. 7 schematisch dargestellt, Doppel-Selektionskassetten konstruiert werden, welche in den gezeigten Kombinationen und Orientierungen das LacZ-Gen bzw. gpt-Gen unter der Kontrolle des 11K-Promotors und/oder des PVEGF exprimieren.In the following, as shown schematically in Fig. 7, double selection cassettes could be constructed which in the combinations and orientations shown express the LacZ gene or gpt gene under the control of the 11K promoter and / or the P VEGF .
Entsprechend Beispiel XX (LacZ-VEGF-Deletion) oder YY (intergenisch-Ba131) lassen sich diese Selektionskassetten nach entsprechender Restriktionsenzymspaltung und Isolierung in die verschiedenen PPV orf DNA-Plasmide inserieren. Nach Insertion einer Doppel-Selektionskassette in das beschriebene Plasmid pdV-500, das eine 312 bp große Deletion des VEGF-Gens von PPV D 1701 aufweist, wurde das Plasmid pMT-10 konstruiert. Durch diese Konstruktion wurden die funktionellen LacZ- und gpt-Gene anstelle des durch die Deletion entfernten VEGF-ORF eingesetzt. Nach transienten Expressionstests ließ sich die Aktivität des LacZ-Gens in PPV D1701-infizierten Zellen beweisen (nicht gezeigt), so daß im weiteren eine Selektion auf gpt- und lacZ-exprimierende VEGF-Deletionsmutanten des D1701 durchgeführt wurde.According to Example XX (LacZ-VEGF deletion) or YY (intergenic Ba131), these selection cassettes can be inserted into the various PPV orf DNA plasmids after appropriate restriction enzyme cleavage and isolation. After insertion of a double selection cassette into the described plasmid pdV-500, which has a 312 bp deletion of the VEGF gene of PPV D 1701, the plasmid pMT-10 was constructed. By this construction, the functional LacZ and gpt genes were substituted for the deleted VEGF ORF. After transient expression assays, the activity of the LacZ gene was demonstrated in PPV D1701-infected cells (not shown) so that further selection was made for gpt- and lacZ-expressing VEGF deletion mutants of the D1701.
Identifizierung oder Schaffung neuer singulärer Restriktionsstellen, die sich in einem intergenischen, nicht-kodierenden Teil befinden. Diese Stellen werden dann verwendet, um fremde DNA-Sequenzen zu inserieren, die funktionelle und nachweisbare Genprodukte (z.B. das LacZ-Gens, von E. coli) oder Teile davon kodieren.Identification or creation of new unique restriction sites located in an intergenic noncoding part. These sites are then used to insert foreign DNA sequences encoding functional and detectable gene products (e.g., the LacZ gene, from E. coli) or portions thereof.
Das Plasmid pORF-PB (Abb. 1) enthält eine einzelne NruI-Schnittstelle, die zwischen der Proteinkinase (F10L) und dem HD1R-Gen liegt. Nach Linearisierung von pORF-PB mit diesem Restriktionsenzym wurde die LacZ-Kassette (nach Auffüllreaktion) 'blunt end' ligiert. Rekombinante Plasmide, die das funktionelle LacZ-Gen enthielten, wurden nach Spaltung z.B. mit BglI selektiert, und die korrekte Insertion durch Southern Blothybridisierung mit einer LacZ-spezifischen Sonde sowie durch partielle DNA-Sequenzierung des Übergangs der LacZ- und PPV-DNA nachgewiesen.The plasmid pORF-PB (Figure 1) contains a single NruI site located between the protein kinase (F10L) and the HD1R gene. After linearization of pORF-PB with this restriction enzyme, the LacZ cassette (after fill-in reaction) was blunt-end ligated. Recombinant plasmids containing the functional LacZ gene were cleaved, e.g. was selected with BglI and the correct insertion demonstrated by Southern blot hybridization with a LacZ-specific probe as well as by partial DNA sequencing of the LacZ and PPV DNA transition.
Die gleiche Vorgehensweise wie im obigen Beispiel beschrieben, wurde stromabwärts des VEGF-Gens (Spaltung an der BstEII-Stelle, Abb. 1) und im potentiellen Gen ORF 3 in der ITR-Region verwendet (partieller Abbau mit XbaI, um eine Spaltung der XbaI-Stelle in den pSPT18-Klonierungsstellen zu verhindern).The same procedure as described in the above example was used downstream of the VEGF gene (cleavage at the BstEII site, Fig. 1) and in the
Um eine neue, singuläre Restriktionsstelle an jeder gewünschten Stelle von klonierten PPV DNA-Fragmenten einzuführen, kann die Technik der PCR-Mutagenese verwendet werden (siehe Abb. 10, Lit. #12). Zu diesem Zweck werden zwei PCR-Reaktionen getrennt durchgeführt, wobei das Primerpaar E1+EV2 (PCR A) bzw. EV1+XB (PCR B) verwendet wird. Alle Primer decken 25 Nukleotide ab, die an den angegebenen Sequenzpositionen mit der PPV-DNA-Sequenz identisch sind. Während Primer XB die authentische Sequenz z.B. um die XbaI-Stelle darstellt (Abb. 1), wurde in Primer E1 am 5'-Ende eine neue EcoRI-Stelle und in die Primer EV1 und EV2 (die komplementär zueinander sind) eine neue EcoRV-Stelle eingeführt. Die EcoRV-Stelle, die in der gesamten Sequenz von pORF-1 oder -2 ursprünglich nicht vorkommt, wurde an die Stelle gebracht, die für die Einführung der LacZ-Cassette gewählt wurde. Die aus den Reaktionen A und B erhaltenen PCR-Produkte werden gereinigt, zu Einzelsträngen denaturiert und unter Reassoziationsbedingungen zusammengemischt; anschließend werden sie für die letzte Reaktion PCR C verwendet. Als Primer werden nun E1 und XB verwendet, um in diesem Beispiel die linken 793 bp von pORF-1 zu verlängern. Nach der Gelisolierung und -reinigung wird das resultierende PCR-Produkt von Reaktion C mit EcoRI und XbaI gespalten und anschließend mit dem durch EcoRI und XbaI gespaltenen Plasmid pORF-XB ligiert. Als Ergebnis enthält das Plasmid pORF-1EV (Abb. 5) die EcoRV-Restriktionsstelle an der gewünschten Position; sie kann dann für eine Linearisierung und Ligation mit der LacZ-Cassette verwendet werden.To introduce a novel, unique restriction site into any desired location of cloned PPV DNA fragments, the technique of PCR mutagenesis can be used (see Fig. 10, ref. # 12). For this purpose, two PCR reactions are carried out separately using the primer pair E1 + EV2 (PCR A) and EV1 + XB (PCR B), respectively. All primers cover 25 nucleotides that are identical to the PPV DNA sequence at the indicated sequence positions. While primer XB contains the authentic sequence, e.g. representing the XbaI site (Figure 1), a new EcoRI site was inserted into primer E1 at the 5 'end and a new EcoRV site was inserted into primers EV1 and EV2 (which are complementary to each other). The EcoRV site, which is absent in the entire sequence of pORF-1 or -2, was placed at the site chosen for the introduction of the LacZ cassette. The PCR products obtained from reactions A and B are purified, denatured to single strands and mixed together under reassociation conditions; then they are used for the last PCR C reaction. As primers E1 and XB are now used to extend in this example the left 793 bp of pORF-1. After gel isolation and purification, the resulting PCR product from reaction C is cleaved with EcoRI and XbaI and then ligated to the EcoRI and XbaI digested plasmid pORF-XB. As a result, the plasmid pORF-1EV (Figure 5) contains the EcoRV restriction site at the desired position; it can then be used for linearization and ligation with the LacZ cassette.
Außerdem können für das in diesem Beispiel beschriebene Verfahren Fehlpaarungs-Primer verwendet werden, die definierte Basenänderungen oder eine Deletion einer einzelnen Base enthalten, um z.B. Translations-Stopkodons oder Aminosäuredeletionen an jeder gewünschten Stelle in der viralen DNA-Sequenz zu erzeugen.In addition, mismatch primers containing defined base changes or a single base deletion may be used for the method described in this example to generate, for example, translation stop codons or amino acid deletions at any desired location in the viral DNA sequence.
In die kodierenden Sequenzen eines der beschriebenen ORFs können nach Spaltung an Restriktionsstellen, die in dem gewählten Gen nur einmal vorkommen, neue oder zusätzliche Sequenzen eingeführt werden.New or additional sequences can be introduced into the coding sequences of one of the described ORFs after cleavage to restriction sites which occur only once in the selected gene.
Die singuläre XcmI-Stelle, die sich im rechten Teil des Gen-Homolog F10L befindet, wurde verwendet, um das Plasmid pORF-1 zu linearisieren. Sowohl die LacZ-Kassette als auch die gespaltene pORF-1-DNA wurden durch T4- oder Klenow-DNA-Polymerase mit glatten Enden versehen, ligiert und kompetente E. coli-Bakterien (DHSαF') wurden für die Transformation verwendet. Die resultierenden Bakterienkolonien wurden durch Kolonie-Filterhybridisierung unter Verwendung einer LacZ-spezifischen Sonde auf positive rekombinante Plasmide getestet und anschließend wurden die entsprechenden Plasmid-DNAs einer Restriktionsspaltung unterzogen.The unique XcmI site, located in the right-hand part of the gene homolog F10L, was used to linearize the plasmid pORF-1. Both the LacZ cassette and the cleaved pORF-1 DNA were blunt-ended by T4 or Klenow DNA polymerase, and competent E. coli bacteria (DHSαF ') were used for transformation. The resulting bacterial colonies were tested for positive recombinant plasmids by colony filter hybridization using a LacZ-specific probe, and then the respective plasmid DNAs were subjected to restriction digestion.
Der VEGF-kodierende Bereich enthält eine einzelne StyI-Stelle, die wie oben beschrieben verwendet wurde, um die LacZ-Kassette zu inserieren.The VEGF coding region contains a single StyI site used as described above to insert the LacZ cassette.
In den folgenden Beispielen wird die Entfernung sowohl codierender (intragenische Deletionen) als auch nicht-kodierender (intergenische Deletionen) Bereiche beschrieben:The following examples describe the removal of both coding (intragenic deletions) and non-coding (intergenic deletions) regions:
Durch Verwendung von Restriktionsenzymen werden definierte Teile des HindIII-DNA-Fragments I entfernt, um die deletierten viralen Sequenzen zu ersetzen, indem man fremde Gene oder Teile davon insertiert. Dies erfolgte durch Spaltung des Plasmids pORF-PA mit dem Restriktionsenzym NruI (an einer Stelle im ITR-Bereich, Abb. 1), wobei ein 396-bp-Fragment deletiert wurde. Nach einer Auffüllreaktion wurde die LacZ-Kassette wie oben beschrieben ligiert. Abbildung 4 zeigt schematisch die Deletion des 396-bp-Fragmentes aus pORF-PA und die Insertion der LacZ-Kassette. Die Abbildungen 5 und 6 zeigen die so konstruierte Deletions-/Insertionsplasmide pCE4 und pCE9 abstammend von pORF-PA.By using restriction enzymes, defined portions of HindIII DNA fragment I are deleted to replace the deleted viral sequences by inserting foreign genes or portions thereof. This was done by cleavage of the plasmid pORF-PA with the restriction enzyme NruI (at a position in the ITR region, Fig. 1), wherein a 396 bp fragment was deleted. After a padding reaction, the LacZ cassette was ligated as described above. Illustration Figure 4 shows schematically the deletion of the 396 bp fragment from pORF-PA and the insertion of the LacZ cassette. Figures 5 and 6 show the thus constructed deletion / insertion plasmids pCE4 and pCE9 derived from pORF-PA.
Einzelne Restriktionsstellen im HindIII-DNA-Fragment wurden als Startpunkte verwendet, um eine bidirektionale Deletion von Sequenzen durch Einwirkung der Endonuclease Bal31 zu bewirken, wie es in Abb. 6 schematisch skizziert ist. Zum Restriktionsabbau wurden beim Plasmid pORF-PA das Enzym StyI und beim Plasmid pORF-1 oder pORF-XB das Enzym XcmI verwendet, um die Gene zu öffnen, die für VEGF bzw. Proteinkinase F10L kodieren (Abb. 1 und 6). Nach Zugabe der Exonuclease Ba131 wurden alle 2 Minuten gleiche Teile der Reaktion entnommen und die Reaktion gestoppt. Spaltung der Zeitwerte z.B. mit dem Restriktionsenzym BglI und anschließende Gelelektrophoresen erlaubten die Abschätzung der Größe des durch Bal31 deletierten DNA-Abschnittes. Gemische von den geeigneten Zeitpunkten wurden dann verwendet, um die DNA-Enden durch eine Auffüllreaktion zu schließen. Zwei komplementäre Oligonukleotide, die neue singuläre SmaI-, SalI- und EcoRV-Restriktionsstellen darstellen, wurden hybridisiert und die resultierenden doppelsträngigen Primermoleküle (als "EcoRV"-Linker bezeichnet) an die mit glatten Enden versehenen Ba131-Produkte ligiert. Nach Transformation von Bakterien wurde Plasmid-DNA isoliert und mit EcoRV gespalten, das in der DNA-Sequenz des PPV-HindIII-Fragments I keine Erkennungsstelle enthält. Daher enthielt jede Plasmid-DNA mit einer EcoRV-Stelle die inserierte Linkersequenz und wurde dann für die Ligation der mit glatten Enden versehenen LacZ-Kassette in die neue EcoRV-Stelle verwendet. Die genaue Größe der erzeugten DNA-Deletion in jeder resultierenden rekombinanten Plasmid-DNA konnte durch Sequenzieren mit den einzelsträngigen EcoRV-Linkern sowie mit geeigneten LacZ-genspezifischen Primern bestimmt werden.Single restriction sites in the HindIII DNA fragment were used as starting points to effect bidirectional deletion of sequences by the action of endonuclease Bal31, as schematically outlined in Figure 6. For restriction digestion, the plasmid pORF-PA used the enzyme StyI and the plasmid pORF-1 or pORF-XB the enzyme XcmI to open the genes which code for VEGF or protein kinase F10L (FIGS. 1 and 6). After addition of exonuclease Ba131, equal parts of the reaction were taken every 2 minutes and the reaction was stopped. Cleavage of the time values e.g. with the restriction enzyme BglI and subsequent gel electrophoresis allowed to estimate the size of the DNA segment deleted by Bal31. Mixtures from the appropriate times were then used to close the DNA ends by a make-up reaction. Two complementary oligonucleotides representing novel SmaI, SalI and EcoRV unique restriction sites were hybridized and the resulting double-stranded primer molecules (termed "EcoRV" linkers) ligated to the blunt-ended Ba131 products. After transformation of bacteria, plasmid DNA was isolated and cleaved with EcoRV containing no recognition site in the DNA sequence of the PPV HindIII fragment I. Therefore, each plasmid DNA with an Eco RV site contained the inserted linker sequence and was then used for ligation of the blunt-ended LacZ cassette into the new Eco RV site. The exact size of the generated DNA deletion in each resulting recombinant plasmid DNA could be determined by sequencing with the single-stranded EcoRV linkers as well as with suitable LacZ gene-specific primers.
Die Kenntnis des DNA-Bereichs, der in D1701 das VEGF kodiert, erlaubt die Herstellung spezifischer DNA-Sonden und PCR-Primer, um dieses Gen in anderen Parapoxvirus-Stämmen zu identifizieren, die äußerst unterschiedliche Restriktionsprofile haben können. Zu diesem Zweck wurden die folgenden Möglichkeiten getestet: (i) Isolierung eines TaqI-Subfragments (366 bp groß) von pORF-PA als Hybridisierungssonde, das den Mittelteil des VEGF-Gens von D1701 darstellt; (ii) Amplifikation des vollständigen VEGF-ORF mit Hilfe geeigneter synthetischer Primer und anschließendes Plasmidklonieren des PCR-Produkts; (iii) Verwendung von Primern, die verschiedene Teile des VEGF-Gens abdecken, wurden für PCRs in Gegenwart von PPV-DNAs als Matrizen sowie als spezifische Hybridisierungssonden verwendet.Knowledge of the DNA region encoding the VEGF in D1701 allows the production of specific DNA probes and PCR primers to identify this gene in other parapoxvirus strains, which may have extremely different restriction profiles. For this purpose were the following options tested: (i) isolation of a TaqI subfragment (366 bp in size) of pORF-PA as a hybridization probe, which is the midportion of the D1701 VEGF gene; (ii) amplification of the complete VEGF ORF using appropriate synthetic primers followed by plasmid cloning of the PCR product; (iii) Use of primers covering various parts of the VEGF gene were used for PCRs in the presence of PPV DNAs as templates as well as specific hybridization probes.
Nach radioaktiver Markierung wurden diese Sonden erfolgreich für Southernsowie Dot/Spot-Blothybridisierung mit der genomischen DNA verschiedener Isolate und Stämme von Parapox ovis, Parapox bovis 1 (Bovines papulöse Stomatitis; BPS) und Parapox bovis 2 (Melker-Knoten) verwendet. Die Southern Blothybridiserung zeigte VEGF-positive Signale mit definierten PPV-DNA Fragmenten, die eine weitere detaillierte Kartierung des VEGF-Gens der verschiedenen PPV erlaubt.Following radioactive labeling, these probes were successfully used for Southern and Dot / Spot blot hybridization with the genomic DNA of various isolates and strains of Parapox ovis, Parapox bovis 1 (Bovine papular stomatitis, BPS) and Parapox bovis 2 (Melker nodules). Southern blot hybridization revealed VEGF-positive signals with defined PPV-DNA fragments, allowing further detailed mapping of the VEGF gene of the various PPV.
Außerdem können dieselben Sonden für vergleichende RNA-Analysen, wie Northern Blothybridisierung, verwendet werden, um die Expression potentieller VEGF-Gene in anderen PPV-Stämmen zu testen.In addition, the same probes can be used for comparative RNA analyzes, such as Northern blot hybridization, to test the expression of potential VEGF genes in other PPV strains.
Konfluent ausgewachsene BK-KL-3A Zellen wurden mit einer Infektionsdosis von 0,1 moi (multiplicity of infection) infiziert. Zwei Stunden später wurden die infizierten Zellen beispielsweise mit DNA (2 bis 10 µg) des Plasmids pMT-10 entweder nach der CaPO4-Glycerolschock-Methode oder unter Verwendung eines Transfektions-Kits nach Angaben des Herstellers (DOSPER, Boehringer-Mannheim) transfiziert. Anschließend wurden diese Zellkulturen mit Selektivmedium (HAT-Medium +MPA Mycophenolsäure - Xanthin - 5 % FKS) während drei bis sechs Tagen bei 37°C, 5 % CO2-Atmosphäre inkubiert, bis cpe oder Plaquebildung sichtbar wurde. Je nach Ausprägung des virus-bedingten cpe wurde:
- (a) Das Zell-Lysat gewonnen, eine Verdünnungsreihe hergestellt und ein Plaque-Test auf BK-KL-3A Zellen durchgeführt. Das zugegebene Agarose-
Mediumgemisch enthielt 0,3 mg/ml Bluo-Gal (Life Science GIBCO-BRL), um blaue Plaques zu identifizieren, die LacZ-exprimierende, MPAresistente D1701-Rekombinanten enthielten. - (b) Nach Bildung von Einzelplaques wurde das in (a) beschriebene Agarose-Bluo Gal-Gemisch zugegeben und blau gefärbte Einzelplaques gepickt.
- (a) Obtain the cell lysate, prepare a dilution series, and perform a plaque assay on BK-KL-3A cells. The added agarose medium mixture contained 0.3 mg / ml Bluo-Gal (Life Science GIBCO-BRL) to identify blue plaques containing LacZ-expressing, MPA-resistant D1701 recombinants.
- (b) After formation of single plaques, the agarose-Bluo Gal mixture described in (a) was added and blue colored single plaques were picked.
Die in (a) oder (b) erhaltenen Viren werden - wie oben beschrieben - zur Infektion von BK-KL-3A eingesetzt und mindestens zwei weiteren Plaquetitrationen und -reinigungen unterworfen, bis eine 100 %ige homogene rekombinante Viruspopulation vorliegt.The viruses obtained in (a) or (b) are used as described above for the infection of BK-KL-3A and subjected to at least two further plaque titrations and purifications until a 100% homogeneous recombinant virus population is present.
Wie in Kapitel 3.1 skizziert, stellt das VEGF-Gen von D1701 ein frühes Gen dar, die spezifische mRNA wird in D1701 virusinfizierten Zellen von zwei bis vier Stunden nach der Infektion an bis zu späteren Zeiten der Infektion in großen Mengen transkribiert. Daher sollte der identifizierte Promotorbereich des D1701-VEGF sehr nützlich für die Steuerung der Expression fremder Gene oder Teile davon in rekombinanten PPV-Viren sein. Die Sequenz, die den VEGF-Promotor umfaßt (35 bis 40 Nucleotide; Sequenz Protokoll ID No: 6), kann isoliert werden durch (i) PCR unter Verwendung der entsprechenden Primer, die den Promotorbereich flankieren, (ii) Subklonieren des entsprechenden DNA-Fragments oder (iii) Synthetisieren der Promotorsequenz als Oligonukleotid. Nach der Verknüpfung des VEGF-Promotors mit jedem interessierenden Gen oder einer DNA-Sequenz kann die resultierende Genkassette zur Herstellung von rekombinantem PPV nach irgendeinem Verfahren, wie es in den vorangehenden Kapitels beschrieben ist, verwendet werden.As outlined in Chapter 3.1, the D1701 VEGF gene is an early gene, the specific mRNA is transcribed in large quantities in D1701 virus-infected cells from two to four hours after infection at later times of infection. Therefore, the identified promoter region of the D1701 VEGF should be very useful for directing the expression of foreign genes or portions thereof in recombinant PPV viruses. The sequence comprising the VEGF promoter (35-40 nucleotides; Sequence Protocol ID No: 6) can be isolated by (i) PCR using the appropriate primers flanking the promoter region, (ii) subcloning the corresponding DNA sequence. Fragments or (iii) synthesizing the promoter sequence as an oligonucleotide. After linking the VEGF promoter to any gene of interest or to a DNA sequence, the resulting gene cassette can be used to produce recombinant PPV by any method as described in the preceding chapter.
Basierend auf der publizierten DNA-Sequenz des 10 kDa Gens des PPV-Stammes NZ-2 (Lit. #8) wurde eine spezifische PCR zum Nachweis des 10kDa-Gens von PPV etabliert. Nach Durchführung der PCR mit den synthetisch hergestellten Primern 10K-up (5-CAATATGGATGAAAATGACGG-3) und 10k-low (5-CAGACGGCAACACAGCG-3) gelang die Amplifikation eines spezifischen, 297 Basenpaar großen Produktes. Die anschließende Klonierung (TA cloning kit, Invitrogen Inc.) ergab das Plasmid pJS-1, welches das 297 bp PCR-Produkt als EcoRI-Fragment enthielt. Die DNA-Sequenzierung beider DNA-Stränge der Insertion von pJS-1 bewies die Anwesenheit der 10kDa-spezifischen Seqzenz. Danach kodiert D1701 für ein 91 Aminosäuren großes 10kDa Protein, das zu dem des PPV-Stammes NZ-2 93,3 % identische bzw. 96,7 % ähnliche Aminosäuren aufweist.Based on the published DNA sequence of the 10 kDa gene of the PPV strain NZ-2 (ref. # 8), a specific PCR for the detection of the 10 kDa gene of PPV was established. After carrying out the PCR with the synthetically prepared primers 10K-up (5-CAATATGGATGAAAATGACGG-3) and 10k-low (5-CAGACGGCAACACAGCG-3), amplification of a specific 297 base pair product was achieved. Subsequent cloning (TA cloning kit, Invitrogen Inc.) yielded plasmid pJS-1, which contained the 297 bp PCR product as EcoRI fragment. DNA sequencing of both DNA strands of the insert of pJS-1 demonstrated the presence of the 10kDa-specific sequence. Thereafter, D1701 encodes a 91 amino acid 10kDa protein that belongs to the of the PPV strain NZ-2 93.3% identical or 96.7% similar amino acids.
Die Lokalisierung des 10kDa-Gens im EcoRI-Fragment E (4,25 kbp) des D1701-Genoms erfolgte durch Southern-Blot-Hybridisierung mit radioaktiv markiertem pJS-1. Demnach ist dieses Gen, ähnlich wie bei NZ-2, im rechten Teil des Virusgenoms lokalisiert und beinhaltet die Schnittstelle der HindIII-Fragmente K und G (Abb. 2).Localization of the 10kDa gene in the EcoRI fragment E (4.25 kbp) of the D1701 genome was by Southern blot hybridization with radiolabelled pJS-1. Thus, similar to NZ-2, this gene is located in the right part of the virus genome and includes the junction of HindIII fragments K and G (Figure 2).
Die Plasmide pDE-E1 und pRZ-E1 die das 4,25 kbp große EcoRI-Fragment E von D1701 enthalten, dienen zur Herstellung von Insertions- und Deletionsplasmiden im 10kDa-Gen (prinzipiell wie vorher beschrieben). Die HindIII-Schnittstelle (Fragmente K-G) im N-terminalen Abschnitt des 10kDa Gens des D1701 (#124-#129 Sequenz ID No: 11) läßt sich sowohl zur direkten Insertion von Fremd-DNA als auch zur Deletion (siehe Ba131 bidirektionaler Verdau) des 10 kDa Gens nutzen. Für letztere Konstruktion wurde die zweite HindIII-Schnittstelle (der multiplen Klonierungsstelle des Vektorplasmids pSPT18) im Plasmid pDE-E1 entfernt. Zu diesem Zweck wurde pSPT18-DNA mit HindIII gespalten, die HindIII-Schnittstelle durch Klenow Behandlung zerstört und religiert. In die EcoRI-Schnittstelle dieses neuen Vektorplasmids pSPT18dH wurde anschließend das 4,25 kbp große EcoRI-Fragment E von D1701 kloniert. Das resultierende Plasmid pRZ-E1 (Abb. 8) besitzt nun eine singuläre HindIII-Schnittstelle im 10 kDa-Gen, welche weitere einfache Manipulation erlaubt.The plasmids pDE-E1 and pRZ-E1 which contain the 4.25 kbp EcoRI fragment E of D1701 are used for the production of insertion and deletion plasmids in the 10 kDa gene (in principle as described above). The HindIII site (fragments KG) in the N-terminal portion of the 10kDa gene of D1701 (# 124- # 129 Sequence ID No: 11) can be used both for direct insertion of foreign DNA and for deletion (see Ba131 bidirectional digestion). of the 10 kDa gene. For the latter construction, the second HindIII site (the multiple cloning site of the vector plasmid pSPT18) was removed in plasmid pDE-E1. For this purpose, pSPT18 DNA was digested with HindIII, the HindIII site disrupted by Klenow treatment and religated. The 4.25 kbp EcoRI fragment E of D1701 was then cloned into the EcoRI site of this new vector plasmid pSPT18dH. The resulting plasmid pRZ-E1 (Figure 8) now has a unique HindIII site in the 10 kDa gene, which allows further simple manipulation.
Southern Blothybridisierung mit pDE-E1 und pJS-1 als radioaktiv markierte Sonden sowie PCR-Untersuchungen zeigen, daß Genome verschiedener PPV bovis 1 keine 10kDa-spezifischen Sequenzen enthalten. Dies weist darauf hin, daß das 10kDa-Gen von PPV nicht essentiell ist (Büttner M. et al. 1996, Lit. #10).Southern blot hybridization with pDE-E1 and pJS-1 as radiolabeled probes, as well as PCR assays, show that genomes of
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Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J. Virol. 62, 1849-1854.16. Falconer, FG and Moss, B. 1988.
Escherichia coli gpt gene provides dominant selection for vaccine virus open reading frame expression vectors. J. Virol. 62, 1849-1854. -
17. Boyle, D.B. and Coupar, B.E. 1988.
Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 10, 343-356.17. Boyle, DB and Coupar, BE 1988.
Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 10, 343-356. -
18. Methods in Virology, Vol. VI, 1977.
edts. Maramorosch, K. and Koprowski H. Academic Press. New York, San Francisco.18. Methods in Virology, Vol. VI, 1977.
Edts. Maramorosch, K. and Koprowski H. Academic Press. New York, San Francisco. -
19. Hattori, M. and Sakaki Y. 1986.
Dideoxy sequencing method using denaturated plasmid templates. Anal. Biochem. 152, 232-238.19. Hattori, M. and Sakaki Y. 1986.
Dideoxy sequencing method using denaturated plasmid templates. Anal. Biochem. 152, 232-238. -
20. Chomczynski, P. and Sacchi, N. 1987.
Single step method of RNA isolation by acid guanidinium-thiocyanatephenol-chloroform extraction. Ana. Biochem. 162, 156-159.20. Chomczynski, P. and Sacchi, N. 1987.
Single step method of RNA isolation by acid guanidinium-thiocyanate phenol-chloroform extraction. Ana. Biochem. 162, 156-159.
Die Sequenz ID No: 1 der Anmeldung zeigt das auf dem HindIII-Fragment I des Stammes PPV D 1701 lokalisierte VEGF-Gen.Sequence ID No: 1 of the application shows the VEGF gene located on HindIII fragment I of strain PPV D1701.
Die Sequenz ID No: 2 der Anmeldung zeigt das auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierte Proteinkinase-Gen F10L (Version 1)Sequence ID No: 2 of the application shows the protein kinase gene F10L (version 1) located on HindIII fragment I of strain PPV D1701
Die Sequenz ID No: 3 der Anmeldung zeigt das auf dem Hindin-Fragment I des Stammes PPV D1701 lokalisierte HD1R-Gessegment.Sequence ID No: 3 of the application shows the HD1R segment located on Hindin fragment I of strain PPV D1701.
Die Sequenz ID No: 4 der Anmeldung zeigt die auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierte ITR-Region und das in dieser Region befindliche ORF3-Gen.Sequence ID No: 4 of the application shows the ITR region located on HindIII fragment I of strain PPV D1701 and the ORF3 gene in this region.
Die Sequenz ID No: 5 der Anmeldung zeigt das auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierte F9L-Genhomolog (Version 1).Sequence ID No: 5 of the application shows the F9L gene homolog located on HindIII fragment I of strain PPV D1701 (version 1).
Die Sequenz ID No: 6 der Anmeldung zeigt die auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierte VEGF-Promotor-Region.Sequence ID No: 6 of the application shows the VEGF promoter region located on HindIII fragment I of strain PPV D1701.
Die Sequenz ID No: 7 der Anmeldung zeigt die auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierte, zwischen dem HD1R- und dem PKF10L-Gen gelegene intergenische Region.
Die Sequenz ID No: 8 der Anmeldung zeigt die vollständige Nukleotidsequenz des HindIII-Fragment I (Version 1) des PPV-Stammes D1701.Sequence ID No: 8 of the application shows the complete nucleotide sequence of HindIII fragment I (version 1) of PPV strain D1701.
Die Sequenz ID No: 9 der Anmeldung zeigt Version 2 des auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierten Proteinkinase F10L-Gens.Sequence ID No: 9 of the application shows
Die Sequenz ID No: 10 der Anmeldung zeigt Version 2 des auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierten F9L-Genhomologs.Sequence ID No: 10 of the application shows
Die Sequenz ID No: 11 der Anmeldung zeigt das auf den EcoRI-Fragment E des Stammes PPV D1701 lokalisierte 10 kDa-Gen.Sequence ID No: 11 of the application shows the 10 kDa gene located on EcoRI fragment E of strain PPV D1701.
Die Sequenz ID No: 12 der Anmeldung zeigt die vollständige Nukleotidsequenz der Version 2 des HindIII-Fragment I des PPV-Stammes D1741.Sequence ID No: 12 of the application shows the complete nucleotide sequence of
Die Sequenz ID No: 13 der Anmeldung zeigt Version 3 des auf dem HindIII-Fragment I des Stammes PPV D1701 lokalisierten Proteinkinase FIOL-Gens.Sequence ID No: 13 of the application shows
Die Sequenz ID No: 14 zeigt die Aminosäuresequenz des PPV D1701 Proteinkinase F10L-Homologs (abgeleitet aus Sequenz ID No: 13).Sequence ID No: 14 shows the amino acid sequence of the PPV D1701 protein kinase F10L homolog (derived from Sequence ID No: 13).
Die Sequenz ID No: 15 zeigt die Aminosäuresequenz des PPV D1701 VEGF-Homologs (abgeleitet aus Sequenz ID No: 1).Sequence ID No: 15 shows the amino acid sequence of the PPV D1701 VEGF homolog (derived from Sequence ID No: 1).
Die Sequenz ID No: 16 zeigt die Aminosäuresequenz des PPV D1701 F9L-Homologs (abgeleitet aus Sequenz ID No: 10).Sequence ID No: 16 shows the amino acid sequence of the PPV D1701 F9L homolog (derived from Sequence ID No: 10).
-
(1) GENERAL INFORMATION:
- (i) APPLICANT:
- (A) NAME: BAYER AG
- (B) STREET: Bayerwerk
- (C) CITY: Leverkusen
- (E) COUNTRY: Germany
- (F) POSTAL CODE (ZIP): D-51368
- (G) TELEPHONE: 0214/3061285
- (H) TELEFAX: 0214/30 3482
- (ii) TITLE OF INVENTION: Parapockenviren die Fremd-DNA enthalten, ihre Herstellung und ihre Verwendung in Impfstoffen
- (iii) NUMBER OF SEQUENCES: 16
- (iv) COMPUTER READABLE FORM:
- (A) MEDIUM TYPE: Floppy disk
- (B) COMPUTER: IBM PC compatible
- (C) OPERATING SYSTEM: PC-DOS/MS-DOS
- (D) SOFTWARE: Patent In Release #1.0, Version #1.30 (EPO)
- (i) APPLICANT:
- (A) NAME: BAYER AG
- (B) STREET: Bayerwerk
- (C) CITY: Leverkusen
- (E) COUNTRY: Germany
- (F) POSTAL CODE (ZIP): D-51368
- (G) TELEPHONE: 0214/3061285
- (H) TELEFAX: 0214/30 3482
- (ii) TITLE OF INVENTION: Parapox viruses containing foreign DNA, their production and their use in vaccines
- (iii) NUMBER OF SEQUENCES: 16
- (iv) COMPUTER READABLE FORM:
- (A) MEDIUM TYPE: Floppy disk
- (B) COMPUTER: IBM PC compatible
- (C) OPERATING SYSTEM: PC-DOS / MS-DOS
- (D) SOFTWARE: Patent In Release # 1.0, Version # 1.30 (EPO)
- (i) APPLICANT:
-
(2) INFORMATION FOR SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 540 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701-VEGF-Gen
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 540 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 VEGF gene
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 2:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1740 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- Proteinkinase-Gen(Version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1740 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 protein kinase gene (version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 3:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1080 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701-HD1R-Genregion
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1080 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701-HD1R gene region
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 4:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1616 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- ITR und ORF3 -Gen
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1616 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 ITR and ORF3 gene
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 5:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 900 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- F9L-Gen (Version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 900 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 F9L Gene (Version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 6:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 94 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- VEGF-Promotor
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 94 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 VEGF promoter
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 7:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 250 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- Intergen. Region zwischen HD1R und Proteinkinase Gen
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 250 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 intergene. Region between HD1R and protein kinase gene
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 8:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 5515 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- HIND III Fragment I (Version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 5515 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701- HIND III fragment I (version 1)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 9:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1620 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 Proteinkinase-Gen F10L (Version2)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1620 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 protein kinase gene F10L (version 2)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 10:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 780 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701-F9L Gen, Version 2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 780 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701-F9L gene,
version 2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 11:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 297 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 10kD- Gen
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 297 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 10kD gene
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 12:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 5519 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D 1701, HindIII-Fragment I, Version 2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 5519 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701, HindIII fragment I,
version 2
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 13:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1742 base pairs
- (B) TYPE: nucleic acid
- (C) STRANDEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D 1701 Proteinkinase-Gen F10L (Version 3)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1742 base pairs
- (B) TYPE: nucleic acid
- (C) BEACHEDNESS: double
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D 1701 protein kinase gene F10L (version 3)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 14:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 497 amino acids
- (B) TYPE: amino acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 Proteinkinase F10L
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 497 amino acids
- (B) TYPE: amino acid
- (C) BEACHEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 protein kinase F10L
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 15:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 132 amino acids
- (B) TYPE: amino acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 VEGF- Protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 132 amino acids
- (B) TYPE: amino acid
- (C) BEACHEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 VEGF protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
- (i) SEQUENCE CHARACTERISTICS:
-
(2) INFORMATION FOR SEQ ID NO: 16:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 224 amino acids
- (B) TYPE: amino acid
- (C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 Protein F9L
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
- (i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 224 amino acids
- (B) TYPE: amino acid
- (C) BEACHEDNESS: single
- (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (iii) HYPOTHETICAL: NO
- (iv) ANTI-SENSE: NO
- (vi) ORIGINAL SOURCE:
- (A) ORGANISM: Parapox ovis
- (B) STRAIN: D1701 protein F9L
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
- (i) SEQUENCE CHARACTERISTICS:
- Abb. 1Fig. 1
- zeigt die physikalische Karte des HindIII-Fragment I aus Orf D1701 in den Plasmiden pORF-1/-2. Die dünnen Pfeile markieren die identifizierten mRNAs, die dicken Pfeile ORFs.shows the physical map of HindIII fragment I from Orf D1701 in plasmids pORF-1 / -2. The thin arrows mark the identified mRNAs, the thick arrows ORFs.
- Abb. 2Fig. 2
- zeigt die physikalische Karte der Hind III Erkennungsstellen auf dem D1701-Genom und die auf dem Hind III-Fragment I identifizierten Gene sowie einen Teil der Inverted Terminal Repeat Region.Figure 4 shows the physical map of Hind III recognition sites on the D1701 genome and the genes identified on Hind III fragment I, as well as a portion of the inverted terminal repeat region.
- Abb. 3Fig. 3
- zeigt Plasmid pCE4. Nach NruI-Spaltung wurde ein 396bp Fragment gegen die LacZ-Kassette ausgetauscht.shows plasmid pCE4. After NruI digestion, a 396bp fragment was replaced with the LacZ cassette.
- Abb. 4Fig. 4
- zeigt Plasmid pCE9 in das nach Linearsierung durch XcmI-Spaltung die LacZ-Kassette inseriert wurde.shows plasmid pCE9 into which, after linearization by XcmI cleavage, the LacZ cassette was inserted.
- Abb. 5Fig. 5
- zeigt schematisch, wie im Text beschrieben, die Strategie zur Erzeugung neuer singulärer Schnittstellen durch PCR.schematically shows, as described in the text, the strategy for generating new singular interfaces by PCR.
- Abb. 6Fig. 6
- zeigt schematisch die bidirektionale Verkürzung durch die Nuklease Bal31 mit anschließender Insertion der EcoRV-Linker und LacZ-Kassette.schematically shows the bidirectional truncation by the nuclease Bal31 with subsequent insertion of the EcoRV linker and LacZ cassette.
- Abb. 7Fig. 7
-
zeigt die Strategie zur Herstellung der LacZ/gpt-Selektionskassette:
- P11K: Vaccinia 11k-Promotor
- PVEGF: PPV VEGF-Promotor
- Sm: Sma I
- B: Bam H1
- P 11K : Vaccinia 11k promoter
- P VEGF : PPV VEGF promoter
- Sm: Sma I.
- B: Bam H1
- Abb. 8Fig. 8
- zeigt schematisch die Klonierungsstrategie des EcoR1-Fragmentes E von PPV D1701, das das 10 kDA Gen enthält (wie im Text beschrieben).Figure 3 shows schematically the cloning strategy of EcoR1 fragment E of PPV D1701 containing the 10 kDa gene (as described in the text).
Claims (4)
- Recombinantly prepared PPVs with insertions and/or deletions, containing a foreign antigen which can induce a pathogen-specific protection.
- Recombinantly prepared PPVs with insertions and/or deletions, containing a cytokine.
- Recombinantly prepared PPVs according to Claim 1 or 2, containing insertions and/or deletions in genome segments which are not required for multiplication of the virus.
- Recombinantly prepared PPVs according to Claim 1 or 2, containing insertions and/or deletions in genome segments which are required for multiplication of the virus.
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SI9730767T SI0886679T1 (en) | 1996-02-28 | 1997-02-17 | Parapoxviruses containing foreign dna, their production and their use in vaccines |
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DE19607458 | 1996-02-28 | ||
DE19607458 | 1996-02-28 | ||
DE19639601A DE19639601A1 (en) | 1996-02-28 | 1996-09-26 | Parapox viruses that contain foreign DNA, their production and their use in vaccines |
DE19639601 | 1996-09-26 | ||
PCT/EP1997/000729 WO1997032029A1 (en) | 1996-02-28 | 1997-02-17 | Parapoxviruses containing foreign dna, their production and their use in vaccines |
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EP (1) | EP0886679B1 (en) |
JP (1) | JP2000507092A (en) |
CN (1) | CN1217027A (en) |
AT (1) | ATE364090T1 (en) |
AU (1) | AU718141B2 (en) |
BR (1) | BR9707785A (en) |
CA (1) | CA2247336C (en) |
DE (1) | DE59712852D1 (en) |
DK (1) | DK0886679T3 (en) |
ES (1) | ES2287949T3 (en) |
HU (1) | HUP9901035A3 (en) |
IL (1) | IL125797A0 (en) |
NO (1) | NO983946L (en) |
NZ (1) | NZ331556A (en) |
PL (1) | PL190401B1 (en) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011135075A1 (en) | 2010-04-29 | 2011-11-03 | Riemser Arzneimittel Ag | Parapoxvirus expressing the vp60 major capsid protein of the rabbit haemorrhagic disease virus |
DE102015111756A1 (en) * | 2015-07-20 | 2017-01-26 | Eberhard Karls Universität Tübingen Medizinische Fakultät | Recombinant Orf virus vector |
Families Citing this family (15)
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KR20000005120A (en) * | 1996-03-29 | 2000-01-25 | 스칸드레트 존 디 | Parapoxvirus vectors |
DK1064382T3 (en) * | 1998-03-17 | 2008-12-08 | Genentech Inc | Homologous polypeptides for VEGF and BMP1 |
WO2000025805A1 (en) * | 1998-11-02 | 2000-05-11 | Ludwig Institute For Cancer Research | Vascular endothelial growth factor-like protein from orf virus nz2 binds and activates mammalian vegf receptor-2 |
WO2002004002A2 (en) * | 2000-07-11 | 2002-01-17 | Bayer Aktiengesellschaft | Use of strains of the parapox ovis virus for producing antiviral pharmaceuticals and anticancer pharmaceuticals |
US6752995B2 (en) * | 2002-04-15 | 2004-06-22 | Board Of Regents, The University Of Texas System | Nucleic acid and polypeptide sequences useful as adjuvants |
EP1499355A4 (en) | 2001-12-07 | 2005-10-05 | Bayer Pharmaceuticals Corp | Use of parapox b2l protein to treat cancer and modify immune responses |
ATE442155T1 (en) * | 2004-07-13 | 2009-09-15 | Aicuris Gmbh & Co Kg | PARAPOX VIRUSES IN COMBINATION WITH OTHER ANTIVIRAL AGENTS FOR THE TREATMENT OF HIV/AIDS |
KR20150015551A (en) * | 2010-05-21 | 2015-02-10 | 조에티스 엘엘씨 | Parapoxvirus vectors containing rabies virus antigen |
MX2013000787A (en) * | 2010-07-20 | 2013-08-08 | Zoetis Llc | Parapoxvirus vectors. |
MX2015000789A (en) | 2012-07-19 | 2015-05-07 | Zoetis Llc | Bovine influenza c virus compositions. |
AU2013295770A1 (en) | 2012-07-27 | 2015-01-29 | Zoetis Services Llc | Tick toxin compositions |
WO2017030901A1 (en) | 2015-08-14 | 2017-02-23 | Zoetis Services Llc | Mycoplasma bovis compositions |
WO2017165366A1 (en) | 2016-03-21 | 2017-09-28 | South Dakota Board Of Regents | Orf virus-based platform for vaccine delivery |
CN112512560A (en) * | 2018-03-07 | 2021-03-16 | 特兰斯吉恩股份有限公司 | Parapoxvirus vectors |
WO2024062098A1 (en) | 2022-09-23 | 2024-03-28 | Transgene | Recombinant pseudocowpox virus encoding an interleukin-12 |
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DE4405841C1 (en) * | 1994-02-23 | 1995-01-05 | Mayr Anton Prof Dr Med Vet Dr | Multipotent non-specific immunity inducers based on combinations of poxvirus components, processes for their preparation and their use as pharmaceuticals |
ES2243937T3 (en) * | 1994-04-29 | 2005-12-01 | Baxter Healthcare S.A. | POXVIRUS RECOMBINED IN ESSENTIAL REGIONS WITH THE HELP OF STRANGE POLINUCLEOTIDES. |
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- 1997-02-17 IL IL12579797A patent/IL125797A0/en unknown
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- 1997-02-17 WO PCT/EP1997/000729 patent/WO1997032029A1/en active IP Right Grant
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- 1997-02-17 CN CN97194139A patent/CN1217027A/en active Pending
- 1997-02-17 AU AU17257/97A patent/AU718141B2/en not_active Ceased
- 1997-02-17 ES ES97904453T patent/ES2287949T3/en not_active Expired - Lifetime
- 1997-02-17 DE DE59712852T patent/DE59712852D1/en not_active Expired - Lifetime
- 1997-02-17 NZ NZ331556A patent/NZ331556A/en unknown
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2011135075A1 (en) | 2010-04-29 | 2011-11-03 | Riemser Arzneimittel Ag | Parapoxvirus expressing the vp60 major capsid protein of the rabbit haemorrhagic disease virus |
DE102015111756A1 (en) * | 2015-07-20 | 2017-01-26 | Eberhard Karls Universität Tübingen Medizinische Fakultät | Recombinant Orf virus vector |
Also Published As
Publication number | Publication date |
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PL328870A1 (en) | 1999-03-01 |
NZ331556A (en) | 2000-05-26 |
CA2247336C (en) | 2008-06-17 |
JP2000507092A (en) | 2000-06-13 |
IL125797A0 (en) | 1999-04-11 |
TR199801702T2 (en) | 1998-12-21 |
AU718141B2 (en) | 2000-04-06 |
AU1725797A (en) | 1997-09-16 |
ES2287949T3 (en) | 2007-12-16 |
CN1217027A (en) | 1999-05-19 |
CA2247336A1 (en) | 1997-09-04 |
SK120098A3 (en) | 1999-03-12 |
HUP9901035A2 (en) | 1999-07-28 |
DK0886679T3 (en) | 2007-09-24 |
EP0886679A1 (en) | 1998-12-30 |
BR9707785A (en) | 1999-07-27 |
HUP9901035A3 (en) | 2001-01-29 |
DE59712852D1 (en) | 2007-07-19 |
ATE364090T1 (en) | 2007-06-15 |
US6365393B1 (en) | 2002-04-02 |
WO1997032029A1 (en) | 1997-09-04 |
PL190401B1 (en) | 2005-12-30 |
NO983946D0 (en) | 1998-08-27 |
NO983946L (en) | 1998-10-22 |
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